Distribution of copper-64 in control mice and in mice bearing ascitic Krebs tumor cells.

Three to 20 hr after an i.p. injection of 64Cu (half-life, 12.8 hr) into mice bearing Krebs ascites cells, a high amount of the radioisotope was recovered in the ascites cells themselves. In the control group, the radioisotope was mainly present in the liver. Similar amounts of 64Cu were recovered in regenerating as well as in normal liver, whereas in the liver of mice bearing ascites cells, this amount was lower by 40 to 50% regardless of the ascitic volume. Thus, the copper metabolism seems to be disturbed at the hepatic level in mice bearing ascites cells. The distribution of 64Cu was 'analyzed in DNA, RNA, and proteins from cellular lysates fractionated by CsCl gradient. There was a uniform pattern of distribution in the macromolecules from ascites cells, while 64Cu' was preferentially associated with the protein fraction from liver. Further experiments indicated that, in vivo, 64Cu was bound to the DNA of ascites cells.

Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment.

PURPOSE: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. METHODS AND MATERIALS: The cell system of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. RESULTS: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10(5) fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. CONCLUSIONS: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumor fragments.

Lack of reverse transformation after treatment of A 549 human alveolar carcinoma cells with thioproline.

Clonal growth in anchorage dependent (on plastic) and independent (in soft agar) conditions of A 549 cells from a human alveolar carcinoma by thiazolidine-4-carboxylic acid (thioproline) was determined in order to check whether this drug was able to induce reversion towards a normal phenotype of in vivo growth, as described by Gosalvez in HeLa cells. The colony formation on plastic, the ability to form three-dimensional clones in soft agar and cell division in mass culture, were inhibited at similar rates when the cells were grown in the presence of increasing concentrations of thioproline. The morphology of cell clones arising on plastic in the presence of thioproline did not differ from that of untreated cell clones. This indicated that thioproline was unable to provoke 'reverse' transformation of A 549 cells in vitro.

Adriamycin uptake and metabolism in organotypic culture of A549 human adenocarcinoma cells according to the exposure time.

In organotypic cultures (nodules) of A 549 human lung adenocarcinoma cells, the long-term cytotoxicity of Adriamycin is strongly improved by shortening the exposure time to the drug. In order to gain insight into the mechanisms of Adriamycin toxicity in this system, we have examined the drug uptake, retention and metabolism by fluorescence microscopy and HPLC analysis. A 549 nodules efficiently metabolize Adriamycin, two major metabolites, adriamycinol and an aglycone derivative, as yet chemically unidentified, are formed and efficiently excreted. Kinetic data show that a long exposure to Adriamycin triggers its efflux from both the nucleus and the cytoplasm while stimulating its metabolism. Therefore, a long exposure time to the drug appears to trigger a process of cellular detoxification by favouring its excretion from the cells via increased metabolism.

A moderate but not total decrease of mitochondrial membrane potential triggers apoptosis in neuron-like cells.

The effects of various degrees of perturbation of the mitochondrial membrane potential (mt delta psi) on apoptosis was investigated by intensified fluorescence digital-imaging microscopy on neuron-like cells, ND7. Mt delta psi was either decreased by 40% by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP 100 nM, 15 min) or completely collapsed (FCCP 10 microM, 60 min). A moderate decrease of mt delta psi induced a reduction of mitochondrial NADH, followed by exposure of phosphatidyl serine and then by chromatin condensation, 36% of nuclei being condensed 60 min after FCCP treatment. During these stages, mitochondrion morphology was fully preserved. In contrast, no chromatin condensation was observed after a rapid and total dissipation of mt delta psi. These results suggest that a partial decrease of mt delta psi would allow mitochondrial functions required to trigger apoptosis to be sustained.

The cytostatic efficiency of gamma-rays on organotypic cultures of malignant cells is greater with irradiation at 23 degrees C compared to 37 degrees C.

The cytostatic efficiency of gamma-rays was determined following irradiation at 23 degrees C or 37 degrees C on A549 human lung carcinoma cells, irradiated either in monolayers or in three-dimensional organotypic cultures (nodules) with a dose-rate of 0.5 Gy/min. For the monolayers, the residual colony forming ability of single cells, and for the nodules, the growth rate, the proportion of regenerating or disaggregating nodules and the growth rate of regenerated nodules were measured. The efficiency of radiation was slightly improved by irradiating the monolayers at 23 degrees C compared to 37 degrees C. The difference was much more pronounced in the nodules, as deduced from (i) length of the time interval between irradiation and appearance of regeneration buds; (ii) growth rate of the regenerated nodules and (iii) growth rate of their progeny analysed over one year of subcultivation. Since organotypic cultures have a structure close to that of tumoral tissue, this result may present some useful applications for tumor radiotherapy.

Cytostatic effects of bleomycin and cis-platinum in A549 human lung tumour cells using autofeeder conditions.

Residual colony forming ability (CFA) of cells from a human alveolar carcinoma (A549) was determined after 48 hours of contact with bleomycin (BLM) or cisdichlorodiammineplatinum (II) (DDP) using feeder cells arising from parallel cultures left in contact with sublethal BLM or DDP concentrations. The CFA of untreated cells was 60 to 80% (45% with X-irradiated feeder cells). Growing cultures were more resistant to DDP and more sensitive to BLM than plateau phase cultures. Subclones arising from a first treatment were resistant to a second BLM or X-ray treatment, but more sensitive to a second DDP treatment.

Growth stimulation by misonidazole of lung carcinoma cells maintained in continuous organotypic and monolayer cultures.

Inasmuch as misonidazole is a drug used in clinical trials for sensitizing radioresistant hypoxic cells in solid tumors, it seemed of interest to study its effects in human tumor cells maintained in tridimensional organotypic cultures. This type of culture involves: spatial organisation of the cells with fairly undisturbed differentiation patterns, minimal traumatizing culture conditions, and offers the possibility to follow post-treatment growth patterns over several months without disturbing the cultures. Misonidazole exhibited a radiosensitizing effect on irradiated nodules derived from a lung adenocarcinoma, and on cells of this tumor growing in monolayers. However, after a 4 hour contact with misonidazole at concentrations corresponding to the range of those found in the serum of treated patients, a significant stimulation of nodule growth was repeatedly observed, together with a strong increase in the frequency of sister chromatid exchanges. Similarly, after treatment of the same tumor cells in confluent monolayers, their colony forming ability was increased. These observations may account for some of the non- convincing therapeutic results obtained in clinical trials.

A single 24h contact time with adriamycin provokes the emergence of resistant cells expressing the Gp 170 protein.

We investigate here, in A549 cells, the influence of a single short (1h) or long (24h) adriamycin (ADR) contact time on the long-term cytotoxicity of the drug and on the emergence of resistant cells. In contrast to a 1 h ADR contact, a 24 h treatment provokes the emergence of resistant cells overexpressing the Gp170 protein and this overexpression is maintained for at least three months without any drug selection pressure.

Altered distribution of copper (64Cu) in tumor-bearing mice and rats.

64Cu was injected in the form of CuCl2 either by subcutaneous or by intraperitoneal route, and its distribution inside different organs was analyzed in 5 different tumor models, 4 in mice and 1 in rats. In all organs tested (blood, liver, kidneys, spleen, intestine, muscle, and tumor) no significant differences were observed in the results obtained after either injection route. All tumors analyzed (Krebs ascite, intestinal Leiomyo sarcoma, human tumor, mammary adenocarcinoma, either spontaneous or chemically induced) contained a relatively high concentration of 64Cu. For all tumor models tested, the 64Cu distribution was altered as compared with that of the corresponding control animals.

Similar lethal effect in mammalian cells for two radioisotopes of copper with different decay schemes, 64Cu and 67Cu.

The decays of 64Cu incorporated in human malignant (A549) or monkey nonmalignant (CVI) cells lead to cell death. When plotted as a function of the radioactivity introduced in the growth medium (microCi/ml at t = 0), the residual colony-forming capability decreases exponentially. The slope of the corresponding curve is steeper for A549 than for CV1 cells. Different data show that the cellular lethal event is a consequence of 64Cu transmutation and not of the irradiation by the simultaneously emitted beta- and beta+ particles. Liquid holding results show that the lethal event is irreparable. The decays of 67Cu, another radioisotope of copper, lead to cell death with the same exponential survival curve and the same lethal efficiency as for 64Cu, in spite of their different decay schemes. The lethal efficiency of both copper isotopes is close to that of 125I utilized in the form of iododeoxyuridine under the same experimental conditions as 64Cu and 67Cu. The high lethal efficiency of radioactive copper transmutations raises questions about the role in DNA functioning of copper atoms known to be present in trace amounts in this macromolecule. The lethal consequence of radioactive copper transmutations suggests that the copper atoms bound to DNA are essential for cellular functioning.

Survival and herpes virus production of normal and xeroderma pigmentosum fibroblasts after treatment with formaldehyde.

The survival of excision-deficient and of excision-proficient (variant) skin fibroblasts from xeroderma pigmentosum (XP) donors was about 5 times and twice, respectively, more sensitive to formaldehyde (FA) treatment than that of skin fibroblasts from healthy and XP heterozygote donors. The capacity of FA-treated host cells to further support Herpes virus (HSV) replication was also more sensitive to FA in XP12BE (group A) than in normal (KD) cells. An important recovery of this capacity occurred in both cell types when they were infected at increasing times (up to 36 h) after FA treatment. This contrasts with the decreasing capacity observed in XP12BE when similarly infected at increasing times after exposure to ultraviolet. In addition, the survival of FA-treated HSV was comparable in KD and XP12BE cells, whereas that of UV-irradiated HSV was much lower in XP12BE than in KD cells.

Survival, DNA synthesis and ribosomal RNA transcription in monkey kidney cells treated by formaldehyde.

Separate cultures of CV-1 cells were exposed for 15 min to 1-16 mM formaldehyde (FA) at various time intervals before labeling with [3H]uridine. The labeled RNA extracted from whole cells was analyzed by polyacrylamide gel electrophoresis. Results indicated that FA produced transcription-terminating lesions in DNA which depressed RNA synthesis, gave rise to shortened RNA chains and modified the expression of transcription-linked genes. These lesions were efficiently repaired since a recovery of RNA transcription, with disappearance of the initially observed alterations, rapidly occurred during post-treatment incubation of cells. Cycloheximide (5 micrograms/ml) strongly inhibited this recovery, whereas fluorodeoxyuridine (10(-5) M) was without effect. The rate of semi-conservative DNA synthesis, measured autoradiographically by thymidine incorporation, as well as the number of cells performing DNA replication, fell to zero after 15-min exposure to FA concentrations greater than 2 mM. Both parameters recovered subnormal levels during a 24-h incubation after treatment with FA concentration up to 8 mM, in agreement with the high survival observed. Unscheduled DNA synthesis was not detectable during the restoration of DNA and RNA synthesis.

Inhibition of DNA synthesis in relation to enhanced survival of UV-damaged herpes virus in monkey cells treated by a variety of 2-nitronaphthofurans.

Various 2-nitronaphthofuran derivatives (related to each other by simple structural modifications) were tested for 2 different effects in CV-1 monkey kidney cell cultures: the immediate inhibition of normal DNA synthesis and the capacity of pretreated cultures (40 h of contact) to support the replication of UV-damaged Herpes simplex virus (HSV). For all compounds tested, a fair correlation was found between their efficiencies to inhibit cellular DNA synthesis and to provoke an increase in UV-HSV production (virus reactivation). Virus reactivation was due to an increase in both the number of virus-producing cells and the amount of infectious particles produced per cell. The most efficient 2-nitronaphthofurans (particularly 2-nitro-7-methoxy-naphtho[2,1-b]furan-R 7000) were at least as potent as aflatoxin B1 in inducing virus reactivation.

Herpes virus production as a marker of repair in ultraviolet irradiated human skin cells of different origin.

When confluent human skin cultures are ultraviolet (UV)-irradiated before infection with Herpes Simplex type 1 virus (HSV), their capacity to support virus growth is impaired. When the time interval between UV-exposure and infection is increased up to 36 hours, different recoveries of HSV production capacity are observed according to the origin of the host cells. 1) Two normal donors: the cells present a dose dependent recovery which is maximal for a dose ( : formula: (see text) at which a plateau level of unscheduled DNA synthesis (UDS) is reached. 2) A mother of two Xeroderma Pigmentosum (XP) children: in this line which exhibits a normal level of UDS, the extent of recovery is significantly decreased after exposures : formula: (see text) 3) An XP child: these cells have a normal level of UDS (XP variant) whereas they present a low extent of recovery as compared with that of the normal subjects. 4) Five XP children: in these excision deficient lines (UDS less than 15%), HSV production capacity decreases with increasing time intervals after UV exposure for doses greater than or equal to 3 : formula: (see text). For doses less than 3 : formula: (see text), a small recovery with an overshoot of viral production is observed 24 h after UV exposure in the lines (three) which present the highest UDS (10--15%) and not in the two lines which present a very low UDS (1--2%).

[Application of the lethal effect of electron capture decay: attempt to control the growth of an ascitic krebs tumor in mice using 64cu]. / Application de l'effet létal de la capture électronique: essai de contrôle par 64cu de la croissance de la tumeur ascitique de Krebs chez la souris.

We have recently demonstrated the existence of a lethal effect due to the transmutation of 64Cu atoms associated with the DNA of in vitro cultured cells: normal monkey cells (kidney) and malignant human cells (alveolar lung carcinoma). The lethal efficiency per decay is high and even higher for the malignant cells. From these results, we have tried to bring about a regression of an ascitic tumor developing in the mouse. The experiments we described show that it is possible to clearly delay (5 X 10(5) ascitic cells injected at t = 0), even to stop for a given number of animals (1 to 2 X 10(4) ascitic cells injected at t = 0), the growth of the tumor owing to some injections of 64Cu totalling about 3 mCi and carried out from the 6th day following inoculation of the malignant cells.

Biochemical studies of human (A549) and simian (CV1) cells labeled with 64Cu, 67Cu, and 125IUdR.

CV1 and A549 cells were grown in the presence of 64Cu porphyric complex, 64CuCl2, or 67CuCl2. Radioactive copper determinations were performed on whole cells and on isolated cellular DNA. 125IUdR was used to calibrate the particular extraction and purification procedures we developed because of the half-lives of 64Cu and 67Cu. The results obtained have shown that some radioactive copper atoms remained firmly bound to the DNA molecule. Their amount was of the same order when using two different DNA isolation methods for the two cell lines studied. No significant differences were found when 64Cu was used as CuCl2 or as porphyric complex.