[CANCER RESEARCH 34, 2969—2974, November 19741

SUMMARY

Radioautographs were prepared from ascites tumor cellsremoved at appropriate time points from tumor-bearing micethat had received bleomycin (BLM)-' 4C in advance. Radioautography indicated LM4 C was adsorbed to the surface ofthe cell membrane of tumor cells at 2 hr. After 4 hr,@ Cwas incorporatedinto the cellsand waslocatedmainlyon thenuclear membrane. There was a sharp increase in thepercentage of necrotic cellsbetween 4 and 8 hr after BLMinjection. Injured cells had densely labeled cytoplasm, thedensity of the label of which gradually decreased withprogressivelyhigher disorganizationafter 8 hr. Therewas noconcentration of grains in the nucleoplasm and little incorporation of isotope into cells in mitosis. It is concluded thatconcentration of silver grains on the nuclear membrane isclosely connected with cell injury caused by this antibiotic.

INTRODUCTION

BLM,' an antitumor antibiotic, was isolated from theculture of Streptomyces verticillus by Umezawa et a!. (22)during the course of a search for antibiotics resemblingphleomycin, which antibiotic is lethal to a wide variety ofbacteria (8), as well as to a variety of mammalian viruses (19)and cells (15). BLM exhibits not only antitumor andantimicrobial activity (2 1, 22) but also is capable of phageinduction (5). Knowledge of the mechanism of action ofphleomycin has been most useful in investigating BLM (20).

BLM causes marked inhibition of DNA synthesis andweaker inhibition of protein synthesis in Escherichia co!i,Ehrlich ascites tumor, and HeLa cells (17). The antibioticlowers the melting temperature of DNA in the presence of asulfhydryl compound (1 1) and also produces strand scission ofDNA both in vitro and in vivo (16, 18). In addition, BLMstimulates degradation of DNA by nuclease and inhibitspolynucleotide ligase (10, 23).

BLM seems to kill selectively noncycling mammalian cells(4) and those during the plateau phase (1). The differentialeffects of BLM on proliferative and nonproliferative cells maybe related to the intracellular distribution of this agent. It waspreviously reported that BLM causes prolongation of survivaltime in mice bearing the Fujimoto tumor (3), as well as muchmore growth inhibition in cultured cells derived from thistumor than in HeLa cells (6). The present study was initiated

I The abbreviation used is: BLM, bleomycin.

Received December 20, 1973; accepted July 17, 1974.

to determine the intracellular distribution of BLM in cells ofthe Fujimoto tumor by means of radioautography.

MATERIALS AND METHODS

Animals and Tumor. C3H/He mice were purchased fromNippon Clear Co., Ltd., Tokyo, Japan. They were maintainedon laboratory chow and were submitted to the experimentwhen their weight was approximately 25 g. A transplantablemouse ascites tumor (Fujimoto) was used. This tumor wasderived from in vitro malignant transformations of C3H/Hemouse embryo cells (3) and is maintained by passing throughthe strain of origin.

Radioautography. 4C, with a specific activity 27.0pCi/mg, was supplied by Nippon Kayaku Co., Ltd., Tokyo,Japan.@ C has an advantage over BLM-3H in providingreproducible data and ensuring stability of the specificactivity. This is related to the position in the BLM molecule ofthe radioactive label: ‘@ C is located at the 3-aminopropyldimethylsulfonum site of this antibiotic.

Twelve days after tumor inoculation, 0.05 ml ascites wasaspirated to serve as a control. Then, BLM.' “C was injectedi.p. at a dose of 1.0 pCi/g body weight (37 pg/g). The drug was

injected within the period of exponential growth of the tumorcells. From each mouse, 0.05 ml ascites was removed at 2, 4and 8 hr after administration of the isotope, and was smearedon glass slides.

Radioautographs were prepared from those smears withSakura NR-M2 emulsion (Konishiroku Photo Industrial Co.,Ltd., Tokyo, Japan) by the modified dry-mounting technique(9, 12) and were exposed for 2 weeks. After developing, slideswere stained with Giemsa.

Morphological Observations. In the same manner as wasused in the radioautography experiments described above,ascites cells were removed from mice into which unlabeledBLM (Nippon Kayaku Co., Ltd.) was injected i.p. at the samedosage. Ascites cells were smeared on glass slides, and theslides were fixed in Carnoy's fixative and stained with Giemsa.

RESULTS

Radioautography. The results of radioautography arepresented in Figs. 1 to 4 and in Table 1. Because adry-mounting technique was used for detection of water-soluble@ C, a rather high background was encountered insome of the radioautographs. The cells with linear arrangementof silver grains on the membrane and/or with a concentrationof grains either in the cytoplasm or in the nucleoplasm werechosen as labeled.

NOVEMBER 1974 2969

Radioautographic Studies on the Intracellular Distribution ofBleomycin-'4C in Mouse Tumor Cells

Jiro Fujimoto

Department ofSurgery, Osaka University Medical School, Osaka 553, Japan

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Cell constituentincorporating@ C%

incorporation after i.p.

injection of@ C atMorphological

appearance2hr 4 hr 8hrCell

membrane4 1631Nuclearmembrane

Cell and nuclear011 6

0 4 14.. .

Withm normallimitmembranesCytoplasm2

3 13NecroticcellsCellswithout uptake94 66 36Consist mostly ofcellsof

@ Cof normal morphologyand of necroticcellsTotal100

100 100

%TimeGeneral

appearancenecroticcellsBefore

injection of BLMThere is considerablevariability in the size ofthe cells and in the sizeand shape of nuclei;some cells have multiplenuclei (Fig.5)3.8After

i.p. injection ofBLM2hrThesameasbefore

injection (Fig.6)7.14hrAppearance of small-sized

basophiic cells(Fig.7)8.28

hrIncrease in number ofnecrotic cells (Fig. 8)25.6

J. Fujimoto

Table 1Pe,centage of the tumor cells incorporating ‘@ C

Two hundred cells were observed per slide of each time point.

In radioautographs prepared from the samples taken fromthe BLM-'4C-injected tumor-bearing mice at 2 hr (Fig. 1),silver grains were seen concentrated on the cell membranes ofthe tumor cells. Cells labeled in this manner constituted 4% ofall of the cells and were of normal morphology. Label waslocalized over the cytoplasm of swelled necrotic cells, whichamounted to 2% of all of the tumor cells. At 4 hr (Fig. 2), inaddition to the greater number of tumor cells with grains onthe cell membrane, grains on the nuclear membrane werenoted, and a few grains were seen in the cytoplasm. These cellswith labeled cytoplasm were necrotic and constituted 3% of allof the tumor cells.

Radioautography at 8 hr (Figs. 3 and 4) revealed an increasein the number of tumor cells incorporating@ C, andespecially a marked increase in the number of necrotic cellswith labeled cytoplasm. At that time, it was observed thatisotope incorporation into the cytoplasm of the necrotic cellsappeared to decrease gradually as the cells became progressively more disorganized.

In the cells in mitosis, label was scarce over the cytoplasmand over the chromosomes, even though grains were concentrated on the cell membrane (Fig. 3). At none of the timesstudied was there a concentration of grains in the nucleoplasm.

Morphological Studies on Tumor Cells Treated with BLM.Morphological studies on tumor cells treated with BLM wereperformed in order to determine whether the tumor cellchanges seen in the radioautography experiments wereattributable to BLM; those changes occurred in a much shortertime and in a higher degree than reported by others (7, 17).The effects of 37 jig BLM per g on the morphology of tumorcells are presented in Table 2 and Figs. 5 to 8. The slides takenfrom BLM injection-treated tumor-bearing mice at 2 hr showfew morphological changes, except for a slight increase innumber of necrotic cells as compared with the control (Figs. 5and 6). At 4 hr, small-sized basophilic cells appeared andnecrotic cells were still slightly increased in number (Fig. 7).At 8 hr, necrotic cells greatly increased in number, reaching amean of 25.6% (Fig. 8).

The mitotic index of the untreated tumor cells was 0.7%.There was no indication at any of these time points that cells

Table 2Effects of 3 7 @igBLM per g on the morphology of the tumor cells

One thousand cells were observed per slide of each time point.

in mitosis decreased in number or received severe injuries as aresult of BLM treatment. These observations are similar tothose on the morphological alterations of the tumor cells bythe radioisotopically labeled drug.

DISCUSSION

A possible interpretation of the intracellular distribution ofBLM is presented in Chart 1. At first, injected BLM isadsorbed to .the surface of the cell membrane of tumor cells.The percentage of BLM-adsorbed cells increases with time.BLM passes through the cell membrane and is incorporatedinto the cells. At 4 hr, BLM is located mainly on the nuclearmembrane. The percentage of cells thus labeled increases withtime. At 8 hr, when numerous tumor cells appear to be injuredby BLM, a large quantity of this drug is localized in thecytoplasm of injured cells. However,@ C incorporationinto the cytoplasm of necrotic cells seems gradually todecrease with progressively greater disorganization of the cells.

CANCER RESEARCH VOL. 342970

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Intracellu!ar Distribution of BLM

seem to be necessarily related to the killing action of BLMitself. The cell membranes of cells that are severely injured byBLM or by other causes may undergo alterations in theirpermeability, with an attendant rush of BLM into thecytoplasm of those cells.

ACKNOWLEDGMENTS

The author expresses appreciation to Dr. H. Higashi and Dr. S. Takaifor their helpful discussion. The labeled and unlabeled BLM was agenerous gift from Nippon Kayaku Co., Ltd., Tokyo, Japan.

2 hr /@hr 8 hr

Chart 1. A scheme for the intracellular distribution of BLM, inmouse tumor cells on the basis of radioautographic analysis. a, a tumorcell of normal morphology; b, a necrotic cell; c, a highly disorganizedcell. For further explanation, see text.

Thus, it seems that once BLM is incorporated into thecytoplasm, it may be released gradually as cells become moredisorganized.

When Ehrlich tumor cells were incubated with BLM in vitro,a 50-fold greater amount of BLM was adsorbed to the surfaceof the cell membrane for S min than was incorporated into thecell for 30 min (24). This was attributed to electrostaticattraction caused by negative charge on the surface of the cellmembrane. The radioautographic findings in this study areconsistent with that report.

The sharp rise in the percentage of necrotic cells between 4and 8 hr after i.p. injection of BLM is thought to be due to cellinjury by this antibiotic (Table 2). Concentration of silvergrains on the nuclear membrane constitutes the salient featureof the radioautograms at 4 hr (Table 1; Fig. 2). Since variousreports have shown that DNA is connected to the nuclearmembrane in the eukaryotic cell (2, 13), BLM incorporationinto the nuclear membrane could result in strand scission ofthe mouse tumor cell DNA (16, 18), in activation of DNase,and in inhibition of its DNA ligase (1 0, 23), postulated as thepossible mode of action of this antibiotic. Such incorporationwould be followed by degradation of DNA and consequentcell injury. Phleomycin, an antibiotic complex produced byanother strain of S. verticil!us (8) and similar to BLM, acts onthe cell wall and membrane of Baci!!us subti!is to induce therelease of membrane-associated DNA and degradation of theDNA (14). The mode of action suggested above for BLM issimilar to these phleomycin findings.

Mitotic cells do not appear to incorporate much@ Cinto their nuclear material (chromosomes), despite concentration of grains on the cell membrane (Fig. 3). It appears thatcells in mitosis neither decrease in number nor receive severeinjuries by BLM treatment (Figs. 5 to 8). These findings maybe regarded as supporting the reports that nonproliferativemammalian cells showed higher sensitivity to BLM thanproliferative ones (1 , 4).

It is consistent with all of the above observations that someof the necrotic cells have labeled cytoplasm. The percentage ofthe cells with labeled cytoplasm is gradually increased inparallel with a progressively higher percentage of necrotic cellswith time after the injection of either labeled or unlabeledBLM (Tables 1 and 2). Therefore, labeled cytoplasm does not

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12. Nagata, T., and Nawa, T. A Modification of Dry-mountingTechnique for Radioautography of Water-soluble Compounds.Histochemie, 7: 370—371,1966.

13. Ormerod, M. G., and Lehmann, A. R. The Release of HighMolecular Weight DNA from a Mammalian Cell (L5 178Y).Attachment of the DNA to the Nuclear Membrane. Biochim.Biophys.Acta,228:331—343,1971.

14. Reiter, h., Milewskiy, M., and Kelley, P. Mode of Action ofPhleomycin on Bacillus subtilis. J. Bacteriol., iii: 586—592,1972.

15. Shuve, S. J., and Rauth, A. M. The Effects of PhleomycinonMouse L-Cells. Cancer Res., 31: 1422—1428, 1971.

16. Suzuki, H., Nagai, K., Akutsu, E., Yamaki, H., Tanaka, N., andUmezawa, H. On the Mechanism of Action of Bleomycin. Strand

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BL M@1@•••TUMORCELL 0 —‘j0 @—@‘

0 hr

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J.Fujimoto

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Fig. 1. Radioautograph prepared from the tumor cells that were removed from a@ C-injected tumor-bearing mouse at 2 hr and stained,after development, with Giemsa. Concentration of silver grains is seen on the cell membrane of the tumor cells. Label is localized over thecytoplasm of the swelled necrotic cell (n). X 400.

Fig. 2. Radioautograph at 4 hr. Concentration ofgrains is noted not only on the cell membrane but also on the nuclear membrane of the tumorcells; n : a necrotic cell with labeled cytoplasm. x 400.

Figs. 3 and 4. Radioautographs at 8 hr, similar to those at 4 hr except for an increase in number of necrotic tumor cells and those incorporating4 C, and a small amount of isotope incorporated into the highly disorganized cells (ii'); m, a cell in mitosis. X 400.

Figs. 5 through8. Smears of Fujimoto tumor cells before and after BLM treatment; the cells were fixed in Carnoy's fixative and stained withGiemsa. Some of these figures represent selected fields of view containing many more cells in mitosis than is typical, in order to show the effects ofBLM on those cells.

Fig. 5. Smear of the tumor cells before treatment. X 400.Fig. 6. Smear of the tumor cells removed from a BLM-injected tumor-bearing mouse at 2 hr. The morphological appearance is the same as before

treatment, except for a slight increase in basophilism of the cells. x 400.Fig. 7 . Smear at 4 hr. Appearance of small-sized basophilic cells is noted. X 400.Fig. 8. Smear at 8 hr. Increase in number of necrotic cells is noted. X 400.

2972 CANCER RESEARCH VOL. 34

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1974;34:2969-2974. Cancer Res   Jiro Fujimoto 

C in Mouse Tumor Cells14Bleomycin-Radioautographic Studies on the Intracellular Distribution of

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