Treatment of mice bearing a Krebs ascitic tumor by means of a protocol based on radioactive copper (64Cu). IV. Consequences of the nature of cancer: tumor-resistant mice.

In the efficient treatment described (Anticancer Research 9: 941-946, 1989) some compounds (64Cu and thioproline) act against the malignant genomes, and other components (metal ions, amino acids, vitamin D2, thyroxine and chelating substances) act against the functioning of the host cells having the cancer functioning (cancer cells). The experiments described show that this treatment developed with mice injected on day zero with 5 x 10(5) Krebs ascitic cells, is also efficient for mice bearing a spontaneous malignant tumor even if it is not an ascitic tumor. After the decrease in weight due to the loss of the tumor, a typical weight evolution was observed characterized by a second weight increase followed by a decrease leading to normal weight. This last weight remained stable. This typical weight evolution in tumor-free mice (after spontaneous tumor or experimental Krebs ascitic tumor) was related to the age of the tumor at the beginning of the treatment, that is, according to our hypothesis (Anticancer Res 9:955-960, 1989) to the cancer evolution emphasizing the great importance of all physiological phenomena related to this cancer course. To test the consequence of this treatment, some of these tumor-free mice were injected with 5 x 10(5) ascitic cells. No tumor developed in these tumor-free mice meaning that they also were tumor-resistant. This last result is in agreement with the forecast of the systemic analysis on which the efficient treatment was based.

Treatment of mice bearing a Krebs ascitic tumor by means of a protocol based on radioactive copper (64Cu). I. The "cancer animal": a new concept leading to an effective antitumoral treatment.

Previous experiments have shown that 64Cu transmutation was inefficient to cure mice which had been injected 6 days earlier with 5 x 10(5) Krebs ascitic cells. The experiments were repeated with 64CuCl2 administered only 1 day after the injection of 5 x 10(5) Krebs ascitic cells when no developing tumor existed. The results reported showed that, even in this case, only a delay in death could be observed. These negative results revealed that malignant cells injected 24 hours previously in a mouse (in vivo conditions) differ from a malignant cell suspension in a tube (in vitro conditions) where the lethal effect of 64Cu transmutation was clearly evidenced. We concluded that some kind of collaboration was established between the few accepted malignant cells and the host. Based on this collaboration we introduced a new concept, "the cancer mouse", in which in addition to the malignant cells some cells in the host, even though non - malignant, nonetheless feature a slightly modified functioning: "the cancer functioning". In this view, an efficient antitumor treatment must act at the same time and in a coordinated manner on the malignant cells and on the host cells which now feature "the cancer functioning". In the efficient treatment described (Anticancer Res 9: 947-954, 1989) some compounds (64Cu and thioproline) act against the malignant genomes, and other components (metal ions, amino acids, vitamin D2, thyroxine and chelating substances) act against the functioning of the host cells by taking into account certain characteristics of living systems. This treatment was efficient in curing mice injected 1 or 6 days previously with 5 x 10(5) Krebs ascitic cells.

Treatment of mice bearing a Krebs ascitic tumor by means of a protocol based on radioactive copper (64Cu). III. Efficiency of the anticancer treatment according to the stage of tumor development.

The treatment described previously (Anticancer Res 9:947-954,1989) was efficient when applied on day 1 or 6 after the injection of 5 x 10(5) Krebs ascitic cells in mice. Under our experimental conditions, all the untreated mice died within 12 to 25 days. The experiments described show that the treatment developed, is efficient when applied on day 11 but not on day 16. To understand the difference in the treatment efficiency between these two days, we tested the 64Cu incorporation inside the ascitic cells. It was observed that 64Cu incorporation exists on day 11 but no longer on day 16. On day 16, the inefficiency of the treatment must be correlated with the non-incorporation of 64Cu inside the ascitic cells. As the tumor growth is also arrested on day 16, an irreversible stage is reached. A model was developed to explain the results obtained. In this model, cancer develops in 3 successive stages. In the first stage, the cellular functioning is under the control of the malignant tumor cells and the number of cells with the "cancer functioning" is increasing with time, but decreasing after removal of the malignant tumor; in the second stage, tumor and cancer both develop independently, meaning that the number of cells with the "cancer functioning" will continue to increase after the removal of the malignant tumor; in the third stage, each cell of the organism has the "cancer functioning" and the characteristics of malignancy will by retroaction.

Treatment of mice bearing a Krebs ascitic tumor by means of a protocol based on radioactive copper (64Cu). II. Analysis of the treatment.

A new treatment is proposed for mice bearing a Krebs ascitic tumor targeting on both the malignant cells and the host cells having the "cancer functioning" (cancer cells) (Anticancer Res 9: 941-946 1989). It is shown that the treatment (a) injure specifically via 64Cu transmutation the DNA of the malignant cells and further perform (with thioproline or spermine) a "reverse transformation" on the damage DNA; (b) restore a "noncancer functioning" in the host cells which had become "cancer cells"; this restoration was performed using, at physiological concentrations, natural compounds already present in all cell types such as metal ions, amino acids, vitamin D2, thyroxine and chelating substances. To decrease the damage induced in the organism by the radiation emitted by 64Cu, a radioprotector, glycerol, was used. Two different strains of mice were used (Swiss or CBA). For each of them, the same treatment was efficient on condition that thioproline or spermine was used for Swiss or CBA mice respectively.

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.

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.

Lethal modifications of DNA via the transmutation of 32P and 33P incorporated in the genome of the S13 bacteriophage.

When circular single-stranded DNA of phage S13 is labelled with 32P or 33P, the transmutations very efficiently bring about a loss of phage infectiousness (efficiency = 1 for 32P and 0.73 for 33P). For both radionuclides, the lethal efficiencies as well as the lethal events are different. In the case of 32P, the lethal event is the loss of the circular integrity of the DNA molecule, occurring as a consequence of a systematic single strand-break caused by each 32P decay (100%). Conversely, in the case of 33P, the lethal events are either a single strand-break (40%) or a local stereochemical modification (33%). The same primary event, the substitution at each 33P decay of a phosphate by a sulfate molecule, leads to one of these lethal events in relation to the decay site. Moreover, neither the phage adsorption nor its genome injection into bacteria depends on the physical state of the genome, and thus lethality is revealed at only the genetic level.

Influence of a growing ascitic tumor in mice on the distribution of injected 65Zn: role of the quantity of zinc administered.

65-Zinc distribution was studied in different organs of either healthy mice or mice bearing an ascitic tumor. This study clearly showed that zinc distribution differs between healthy and tumoral mice. Moreover, zinc distribution in the different organs of the mice is modified by the quantity administered.

Differences in the consequences of the decay of radioactive 64Cu atoms incorporated in cells under either in vitro or in vivo conditions.

To study the lethal effect of 64Cu under in vitro conditions, asynchronous mammalian cells were used. A lethal effect does exist as a consequence of the decay itself of a few 64Cu atoms incorporated in cellular DNA. This lethal effect is characterized by an exponential survival curve with no shoulder and no tail; it exists even for non-dividing cells. The lethal efficiency per decay is very high. To test the 64Cu lethal effect under in vivo conditions, experiments were performed with ascitic cells developing in mice. In this case, the lethal effect also exists, but it is not a function of the 64Cu doses injected in the mice. Faced with this puzzling result, a systemic approach was necessary to understand and counteract ascitic cells developing in mice.

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.

[Lethal effect of the decay of 64Cu and 67Cu incorporated in mammalian cells]. / Effet létal de la désintégration de 64Cu et de 67Cu incorporés dans des cellules de mammifères.

A high lethal efficiency was observed when the decay of two radioactive isotopes of copper, 64Cu or 67Cu, occurred in mammalian cells. The lethal efficiency is of the same order for both isotopes in spite of their different decay processes. As the lethal event could be attributed to an injury inside the cellular DNA only, these results suggest that, even if present in the DNA in trace amounts solely, copper atoms are essential chromatin components. Their behaviour differs depending on whether the cell is a tumor or a non tumor cell because their lethal efficiencies are different (0.5 and 0.1 respectively).

[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.

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.

Implications of reiterative DNA-Metal ion complexes in the induction and development of neoplastic cells.

Experimental data on the content in metal ions of DNA preparations from various neoplastic and healthy tissues are summarized: metal ions are preferentially bound to reiterative DNA sequences, where they may induce conformational variations and thus modify the binding of effector molecules such as repressors and polymerases.A model is described where essential and toxic metals are successively loaded on ligand acceptor groups of increasing affinity and thus may reach the final active sites: enzymes and reiterative DNA sequences (equated at least partially to regulative DNA sequences). The effects of some molecules, including peptides, antibiotics, growth factors, hormones, and antineoplastic substances, on DNA conformation could be explained in part by their chelating ability.The neoplastic state may be induced by a modification of metal ion transfer chains: quantitatively by a continuous derepression of genes coding for metal ligands, genes that are only temporarily derepressed during development in normal cells, and qualitatively by modifications of the nucleotidic sequence of structural genes leading to an increase of the chelating ability of the coded metal ligand.

[Preferential localization of cadmium on the iterative DNA sequences from cultured tissues of the crown gall of tobacco (Nicotiana tabacum, var. Wisconsis 38)]. / Localisation préfèrentielle du cadmium sur des séquences itératives de l'ADN isolé de cultures de tissus de crown-gall de tabac (Nicotiana tabacum, var. Wisconsin 38)

Fractionation of total crown-gall tissue culture DNA from Nicotiana tabacum by Ag+-Cs2SO4 density gradient is described. Cadmium ions determination is performed in each fraction by anodic stripping voltammetry. The cadmium content of the DNA in the lightest density fractions is 100 to 1000 times higher than in the other fractions.