Bleomycin: mammalian cell lethality and cellular basis of optimal schedule.

Experiments on the dose- and time-dependent changes in the survival of Ehrlich ascites tumor cells exposed to bleomycin were done to determine a useful regimen for the effective inactivation of the tumor cells. The experimental results on time-dependent changes in survival of bleomycin-treated cells indicated that two phenomena were involved in the survival increase observed after single bleomycin treatment: 1) The bleomycin-treated cells were resistant to the second injection when the interval between the two successive doses was within 2 hours (induced resistance), and 2) the survival was increased as a function of time when the interval between treatments was prolonged (repair of potentially lethal damage). The effect of the fractionated treatments was then investigated for the regimen that minimized the induced resistance and repair of potentially lethal damage. The results indicated that administration of a lower dose of bleomycin at shorter intervals was more effective than other fractionation schedules, and, on the basis of the same dose rate, the continuous infusion regimen was more effective than fractionation regimens for the sterilization of Ehrlich ascites tumor cells.

The synthesis of Forssman glycolipid in clones of nil 2 hamsters fibroblasts grown in monolayer or spinner culture.

The amount of Forssman glycolipid (GL-5) was investigated in two clones of Nil hamster cells grown either in monolayer or in spinner culture. GL-5 assayed by the complement-fixation inhibition test increased with increasing cell density in the monolayer. However, cells grown in spinner cultures failed to show the density-dependent response in both clones examined. Cells transformed by hamster sarcoma virus did not show the density-dependent increase of GL-5, even in cells grown in monolayer. The effect of transfer from confluent to sparse cultures on the amount and the synthesis of GL-5 was also examined. It is suggested that the GL-5 that accumulates in cells during confluency is diluted into the daughter cells and that the decrease of the Forssman lipid does not precede cell division.

Inhibition of X-ray or chemical carcinogen-induced neoplastic transformation of C3H10T1/2 fibroblasts by lipopolysaccharides.

Oncogenic transformation of mouse 10T 1/2 fibroblasts induced upon exposure to X-ray or N-methyl-N'-nitro-N-nitrosoguanidine was suppressed if lipopolysaccharide (LPS) was present in the culture medium. The suppressive effect of LPS was exerted within 24 h after irradiation. Suppression was dependent on the concentration of LPS added and LPS (2 micrograms/ml) derived from Salmonella minnesota R595 reduced the number of transformed type III foci per dish from 0.39 to 0.15. Indomethacin (1 to 30 microM) further enhanced the effect of LPS in a dose-dependent manner.

Immune elimination of host fibroblasts for the cultivation of human tumors transplanted into nude mice.

We report here a useful method for the isolation and cultivation of human tumor cells in vitro from human tumors grown in nude mice. A rabbit was immunized with spleen cells obtained from adult nude mice. The rabbit antiserum in the presence of complement effectively killed cultured cells derived from various mouse tissues, but it was not cytotoxic to cultured cells from human tissues including tumors. When mixed cultures consisting of human tumor cells and nude mouse fibroblasts were treated with the antiserum and complement, the nude mouse fibroblasts were completely removed from the cultures, and the human tumor cells could be propagated without noticeable changes in morphological features. Primary cultures of heterotransplanted human tumors grown in nude mice were also successfully treated, resulting in the ultimate elimination of fibroblastic cells derived from the stroma of the tumor. The functional properties of the tumor cells (production of human chorionic gonadotropin by choriocarcinoma cells and production of carcinoembryonic antigens by pancreas carcinoma cells) were also maintained after the antiserum treatment.

Enhancement of mammalian cell killing by 5-fluorouracil in combination with X-rays.

The enhanced cytocidal effect of a combination of X-rays and 5-fluorouracil was investigated by means of colony-forming ability in mouse L-cells. Cells were treated with 5-fluorouracil immediately preceding (preirradiation treatment) or following (postirradiation treatment) irradiation. In either pre- or postirradiation treatment with various concentrations of the drug for a fixed time, the enhanced effect was augmented with increasing concentrations of 5-fluorouracil up to 20 microgram/ml. When cells were subjected to postirradiation treatment with a fixed concentration of drug for varying times, the cytocidal effect was further enhanced with increasing duration of drug treatment. In preirradiation treatment, however, drug treatment for longer than 3 hr did not exhibit any further enhancement. Postirradiation treatment with 5-fluorouracil to synchronous cells at various ages demonstrated enhancement at all ages during the cell cycle. The greatest enhancement was observed in the DNA-synthetic phase. Postirradiation treatment with 5-fluorouracil (2.5 microgram/ml) for 24 hr markedly reduced the width of the shoulder of the X-ray survival curve without significantly altering the slope of the exponential portion of the curve. Recovery from sublethal radiation damage was not suppressed by 5-fluorouracil when cells were treated with drug between fractionated X-ray doses. These results indicate that damages caused by 5-fluorouracil and X-rays interact additively to induce cell killing.

Neoplastic transformation of plateau-phase mouse 10T1/2 cells following single and fractionated doses of X rays.

The induction of cell transformation and cell killing in plateau-phase 10T1/2 mouse cells by single and fractionated doses of X rays was investigated. The single dose-transformation curve was composed of a first phase (doubling dose = 0.2 Gy), a second phase (doubling dose = 1 Gy), and a third phase (no increase of transformation at doses more than 6 Gy). Two-dose fractionation experiments revealed that transformation damage was reduced when total doses of 0.93, 1.86, and 3.72 Gy were given with two equal fractions separated by intervals of 3 to 15 hr, as compared with the equivalent single doses. This not only represents repair of subtransformation damage but also is consistent with previous findings of repair of potential transformation damage. The fractionated dose-transformation curve obtained 3 hr after the first (conditioning) dose indicated that damaged cells had recovered, at least in part, from transformation damage, as shown by the reappearance of the first phase of transformation induction. No alteration of sensitivity to the second dose seemed to develop; however, the transformable fraction of the cell population was apparently increased by the conditioning dose. From the present results with single and fractionated doses, a resolution between repair of sub- and potential transformation damage did not seem possible. Plateau-phase cells were found to be useful for dose fractionation studies.

Radiation-induced neoplastic transformation of C3H10T1/2 cells is suppressed by ascorbic acid.

X-ray induced transformation of C3H10T1/2 cells was suppressed in a concentration-dependent manner by administration of ascorbic acid after irradiation (0.1-20 micrograms/ml for the first week) in the culture medium. The dose-response curve was shifted about 60% downward and was slightly steeper in the presence of ascorbic acid (5 micrograms/ml for the first week) than in its absence. The 1-week treatment procedure revealed that cells initiated by radiation remained susceptible to ascorbic acid until the time of morphological phenotype expression. The neoplastically transformed phenotype expressed after incubation for 8 weeks could no longer be suppressed by ascorbic acid even after culture transfer. Similarly, the neoplastically transformed phenotype suppressed for 8 weeks by ascorbic acid treatment was not subsequently expressed in the absence of ascorbic acid. On the basis of the oxygen-detoxifying nature of ascorbic acid, we postulated that expression of the neoplastically transformed phenotype is promoted by reactive oxygen species and peroxy radicals generated in cells during the whole assay period. The data may be useful as a guide for chemopreventive efforts against radiation carcinogenesis.

Induction of malignant transformation in mouse 10T1/2 cells by low-dose-rate exposure to tritiated water and gamma-rays at two different temperatures, 4 degrees C and 37 degrees C.

Cultured mouse (C3H 10T1/2) cells in contact-inhibited state were subjected to protracted exposure at 4 degrees C or 37 degrees C to either beta-rays from HTO or 60Co gamma-rays. The duration of exposure was 20 h and the total dose was varied by changing the dose-rate. The dose-survival and dose-transformation curves for gamma-irradiation at 4 degrees C were close to those obtained after a single acute X-ray dose (0.5 Gy/min). When gamma-irradiation was administered at 37 degrees C, both the lethality and transformation induction were lower than those after the corresponding doses at 4 degrees C, presumably owing to repair of lethal and transformational damage during gamma-irradiation at 37 degrees C. The same effect of temperature was observed in the case of HTO exposure, indicating again the existence of repair of damage during beta-irradiation at 37 degrees C but not at 4 degrees C. These facts strongly suggest that when irradiated at a low temperature such as 4 degrees C, both the dose-lethality and dose-transformation induction relationships were independent of the dose-rate for either gamma- or beta-exposure, at least the dose-rates used in this experiment. Thus, the comparison of radiation effectiveness between beta- and gamma-exposures at 4 degrees C gave reliable values of relative biological effectiveness (RBE) of tritium beta-rays which were ca. 1.4 at the D0 for lethality and 1.6 for cell transformation within the dose range examined (1 to 6 Gy of beta-rays). The comparison between exposures at 37 degrees C resulted in RBE values (1.4 and 1.7, respectively) very close to those at 4 degrees C.

Cell cycle dependent response and combined treatment of tumor.

Attempt was made to summarize data indicating cell cycle dependence of survival response to various antitumor agents. Normalized patterns of survival response permitted us to group agents into two major categories. It was known that effect of agents on cell progression was not only related to respective periods of the cell cycle but also rather specific for each category of agents. The extent of progression inhibition was not compared on a quantitative basis. On the basis of above findings a rational combination and a scheduled administration of agents for effective sterilization of tumor cells were considered.

[Lethal effect of bleomycin on Burkitt lymphoma cells at stationary phase].

Effects of bleomycin on cultured Burkitt lymphoma cells (P3HR-1) at the stationary phase were studied. They showed a marked less sensitivity to bleomycin than the cells at the exponential phase. The cell kinetic analysis revealed that the stationary phase was occupied by G1 cells, which were different from exponential G1 cells at various points of metabolic and nutritive state except for DNA content. Then, by synchronizing the exponential cells with hydroxyurea, the response of G1 cells to bleomycin was obtained. As a result, their sensitivity to the drug was found to almost coincide with that of the stationary cells. Therefore, the less sensitivity of the stationary cells to bleomycin was due to the total occupation of the stationary phase by G1 cells showing originally resistance to bleomycin.