`
`(1981)
`
`Cell Surface Charge and Cell Division in Escherichia coli
`after X Irradiation
`
`CHIKAKO SATO,* KIYOHIDE KOJIMA,. KIMIKO NISHIZAWA,*
`AND YUKINORI HIROTA$
`
`*Laboratory of Experimental Radiology, Aichi Cancer Center Research Institute,
`Chikusa-ku, Nagoya 464, Japan
`tLaboratory of Oncological Pathology, Nara Medical University, Kashihara 634, Japan
`tLaboratory of National Institute of Genetics, Mishima 411, Japan
`
`SATO, C., KOJIMA, K., NISHIZAWA, K., AND HIROTA, Y. Cell Surface Charge and Cell
`Division in Escherichia coli after X Irradiation. Radiat. Res. 87, 646-656 (1981).
`
`Simultaneous detection of electrophoretic mobility (EPM) and morphology of individual
`irradiated Escherichia coli cells under the phase microscope revealed a concurrent decrease
`in EPM and arrest of cell division. EPM decreased with time and reached a minimum 15 min
`after irradiation with doses ranging from 100 R to 80 kR. Cells elongating due to the division
`block retained the minimum EPM. After a recovery phase, separated small-sized daughter
`cells and some long filamentous cells, which had a few cleavages at the termini, returned to
`the normal EPM. This finding indicates that recovery in EPM, which represents recovery in
`the surface architecture, precedes or coincides with the resumption of cell division. Nuclear
`staining of the recovering cells leads to the suggestion that the cleavage of the cell takes place
`whenever the EPM has recovered, irrespective of the segregation of DNA, which gives rise
`to anuclear cells having normal EPM. It is suggested that the mechanism of EPM decrease
`is Ca2+-dependent conformational change of the membrane accompanying vertical translo-
`cation of charged groups.
`
`We have investigated the role of membrane damage in impaired proliferation
`and death of cells after X irradiation using cell electrophoresis to detect membrane
`damage. The advantages of this method are its sensitivity as a probe to detect the
`alteration of dynamic physiological charge-related properties on the surface of
`living cells (1-3), and the simultaneous detection of the electrophoretic mobility
`(EPM) and the morphology of individual cells under the phase microscope. Esch-
`erichia coli was used as a model system to study the relationship between cell
`division and the cell surface. Bacteria inhibited in cell division are easily seen during
`measurement of EPM because of their filamentous form. Decrease in EPM after
`irradiation has been reported in several types of mammalian cells and yeasts (4-
`6). Our previous work on three lines of cultured mammalian cells revealed that the
`fraction of cells whose EPM did not recover within 24 hr after irradiation was
`statistically in agreement with the fraction of non-colony-forming cells (7-9). The
`present experiments utilizing E. coli clearly indicated that EPM decreased in cells
`when cell division was inhibited, and that recovery of EPM occurred at the re-
`sumption of the cell division.
`
`0033-7587/81/090646-11 $02.00/0
`Copyright ? 1981 by Academic Press, Inc.
`All rights of reproduction in any form reserved.
`
`646
`
`Radiation Research Society
`is collaborating with JSTOR to digitize, preserve, and extend access to
`Radiation Research
`®
`www.jstor.org
`
`Page 1 of 11
`
`HOLOGIC EXHIBIT 1034
`Hologic v. Enzo
`
`
`
`SURFACE CHARGE AND CELL DIVISION
`
`647
`
`MATERIALS AND METHODS
`
`Cells and culture. An Escherichia coli K-12 strain, PA3092 (Fthr-leu-lacy-
`trp-his-thy-str-malA-xyl-mtl-arg-suII-), was used. The cells were cultured with
`aeration in L-tubes at 37?C in L broth (1% bactotrypton, 0.5% yeast extract, 0.5%
`NaCI, 0.1% glucose) supplemented by 50 mg/liter thymine. Only cells growing
`exponentially with the shortest doubling time were used. The cell maintains a
`constant electrophoretic mobility during the logarithmic phase of growth but shows
`a reduced mobility at the stationary phase.
`Irradiation. X irradiation of the cells was carried out in culture medium in a
`3-cm-diameter plastic Petri dish (2-mm depth of cell suspension) at 3?C on ice.
`The physical factors of exposure were: 200 kVp, 20 mA, 0.5 mm Al + 0.5 mm Cu
`filter added, half-value layer 1.13 mm Cu, 25-cm target-sample distance, and
`exposure rate in air 425 R/min.
`Electrophoresis. At different times of incubation in shaking L-tubes at 37?C
`after irradiation, an aliquot of cell suspension was placed on ice, centrifuged, and
`then washed in cold 67 mM phosphate buffer supplemented with 5.4% sorbitol for
`electrophoresis. The electrophoretic mobility of individual cells was measured at
`25 ? 0.5?C with a Zeiss cytopherometer as reported in (5, 10, 11). Each cell was
`allowed to move 16 ,m in a scaled thin chamber under the phase microscope
`alternatively in both directions following reversal of current (4 mA) in the 67 mM
`phosphate buffer supplemented with 5.4% sorbitol. The 67 mM phosphate buffer
`(pH 7.3) contained 50.1 mM Na2HPO4 and 16.5 mM KH2PO4. The ionic strength
`and osmolarity of the buffer were usually indicated as 0.167 and 183 mosm with
`the assumption of complete dissociation of phosphates. However, the conductivity
`of the buffer supplemented with 5.4% sorbitol was 7.471 X 103 #U3/cm. This value
`was about one-half the conductivity of the 167 mM NaCI solution, thus indicating
`about 50% dissociation of phosphates. Since 5.4% sorbitol is isotonic (about 300
`mosm), the electrophoresis medium is hypertonic by about 183/2 mosm due to
`phosphates. For the measurement of mobility at lower ionic strengths of solution,
`phosphate buffers diluted stepwise (6.7, 13.4, 26.8, 40.2, 53.6 mM) were supple-
`mented with 5.4% sorbitol to maintain the same viscosity. The conductivities of
`the medium were 1.076 X 103, 1.811 X 103, 3.413 X 103, 4.784 X 103, and 6.141
`X 103 uL3/cm, respectively. Osmolarity of the medium varied from about 309 to
`373 mosm. We chose the same concentration of sorbitol because EPM is dependent
`on the ionic strength and the viscosity of the medium, but not on its osmolarity.
`The mobility was determined from separate experiments on 10-100 cells for each
`set of conditions and calculated as tzm-sec
`.V-' .cm.
`Morphological observations. Cells were fixed in 10% formalin for 5 min, washed
`with phosphate-buffered saline, and then spread on a glass slide coated with poly-
`L-lysine using a cytocentrifuge (Shandon Elliott) for 5 min at 1500 rpm. The cells
`were dried on the slide, treated with 1 N HC1 for 5 min at 60?C to digest ribonucleic
`acid, washed by running water, and then stained in freshly diluted Giemsa solution
`(Merck). Distribution of chromosomes and the size of the cells were detected using
`a microscopic photograph. The cells in the suspension were counted with a he-
`mocytometer after different incubation periods.
`
`Page 2 of 11
`
`
`
`648
`
`SATO ET AL.
`
`RESULTS
`
`Morphological change after irradiation. Figure 1 shows the nuclear staining of
`cells indicating changes in cell size and distribution of nuclear mass with time at
`37?C after irradiation with 50 kR. Nonirradiated cells (Fig. 1A) were about 2.5
`,m in mean length and contained one to four nuclei per cell. The cells with one
`nuclear mass were only 3.2% of the cells in nonirradiated culture, but were 47 and
`91% (Fig. iB) of the cells 15 and 30 min, respectively, after irradiation with 50
`kR. As shown in Fig. 2, the mean number of nuclear masses in a cell decreased
`from 2.86 to 1.11 during the first 30 min of incubation after the exposure. During
`this 30 min, the number of cells increased about 1.6-fold, and the increase in cell
`length was slight. These results suggest that cell separation proceeded in those cells
`in which the chromosomes had segregated before irradiation. Enlargement of con-
`densed nuclear masses at the central site of cells and the elongation of the cell
`progressed after 30 min without further separation of the cells (Figs. 1C, D). A
`cleavage of the cell was noticed at the central portion of some condensed nuclei.
`Morphological disintegration of cells appeared only after 2 hr of incubation when
`cell division resumed. At 3 hr of incubation, cell division was often observed in the
`filamentous cells in which chromosomes had been segregated to the daughter cells
`as shown by arrows in Fig. 1E. The filaments with a condensed nucleus at the
`center on occasion caused cell separation from the termini, thereby giving rise to
`anucleated cells. During the next 2 hr, normal-sized cells proliferated and became
`predominant over lysed cells and long filaments (Fig. 1F).
`Change in electrophoretic mobility (EPM) with time after irradiation. Figure
`3 shows the time course of change in EPM after irradiation with different doses.
`Decrease in EPM was detectable even after irradiation with 100 or 500 R, but it
`returned to normal rapidly during the subsequent incubation for 15 or 45 min,
`respectively. The EPM reduction was not different and was maximum 15 min after
`irradiation with doses ranging from 15 to 80 kR. EPM recovery, however, was
`dependent on dose; recovery began earlier and reached a higher value after smaller
`doses of irradiation. Comparison of the time course change in cell length and EPM
`indicated that EPM recovery began 2 hr after irradiation with 50 kR when the
`length of the cell was maximum.
`Frequency distribution of EPM and cell size. Figure 4 illustrates the frequency
`distribution of EPM of unirradiated cells and of cells exposed 30 min earlier to 50
`kR. Every irradiated cell showed reduced EPM, and the two distributions are
`clearly separated with the boundary at -1.2 gm. sec-' V-' -cm. The cells irradiated
`with 50 kR progressively elongated to form filaments with incubation periods up
`to 2 hr and kept the minimum EPM. Thereafter separation of normal-sized cells
`took place from the termini of the long filaments as shown in Fig. 1. Three hours
`after irradiation with 50 kR, about 31% of the cells were of small size (below 4
`Am). Figure 5 exhibits the relationship between EPM and the length of the cell.
`The distribution of the points is separated into three groups. All the short cells
`(below 4 ,um) had EPM higher than -1.2
`tm-sec-' V-' .cm as nonirradiated cells.
`EPM of the longer cells distributed mostly below -1.2, but 30% of them showed
`
`Page 3 of 11
`
`
`
`SURFACE CHARGE AND CELL DIVISION
`
`649
`
`.-?~ 0. . <j
`
`,.0
`
`BE_ iii
`
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`
`FIG. I. Nuclear staining of Escherichia coli K- 12 indicating cell size and distribution of chromosomes.
`Cells were fixed with 10% formalin at 30 min (B) or I (C), 2 (D), 3 (E), or 5 hr (F) after irradiation
`with 50 kR, treated with I N HC1 for 5 min at 600C, and then stained in Giemsa solution. Arrows in
`(E) indicate cleavages of elongated cells and an anuclear cell. (A) Nonirradiated cells. Bar in (A) is
`10 um.
`
`Page 4 of 11
`
`
`
`650
`
`SATO ET AL.
`
`E
`c
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`Incubation
`
`15
`30
`time after
`
`irradiation
`
`60
`(min)
`FIG. 2. Changes in (O) cell length (jIm), (X) cell density (X107 cells/ml), and (*) number of nuclei/
`cell with incubation time after irradiation with 50 kR. Each point is the mean value of measurements
`on more than 400 cells. Results suggest early separation of cells in which the nuclei had previously
`segregated at the time of irradiation, and the later elongation of cells without nuclear segregation and
`cell division.
`
`the higher EPM. A few cleavages per cell were often noticed in those filamentous
`cells having EPM higher than that of the ordinally filamentous cells. Closed circles
`in Fig. 5 represent those filamentous cells containing visible cleavage, which always
`showed EPM greater than -1.2. Morphology of these cells is exhibited in Fig. 1E.
`About 10% of elongated cells without visible cleavage also showed EPM above
`
`0 15 30
`60
`120
`180
`Time after
`X-irradiation(min)
`FIG. 3. Change in electrophoretic mobility (- m-.sec-'.V-'.cm)
`of E. coli with incubation time at
`37?C after irradiation with 100 R (0), 500 R (A), 15 kR (0), 30 kR (A), 50 kR (*), or 80 kR (X).
`Each point represents the mean value of measurements on more than 30 cells from three separate
`experiments.
`
`37?C
`
`240
`
`Page 5 of 11
`
`
`
`SURFACE CHARGE AND CELL DIVISION
`
`651
`
`50 kR 30min
`
`OR
`
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`1.2
`1.0
`ic mobility
`Electrophoret
`FIG. 4. Frequency distribution of electrophoretic mobility (-m
`-sec-'. V-' .cm) of unirradiated cells
`(shaded columns) and cells exposed to 50 kR 30 min earlier (open columns).
`
`-1.2. These results suggest that the recovery of EPM precedes or coincides with
`the separation of the daughter cells.
`Effect of ionic strength on mobility. EPM was measured in a buffer solution of
`
`0
`
`35.
`
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`
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`
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`
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`
`1.5
`
`1.6
`
`Electrophoretic
`mobility
`FIG. 5. Relationship between the electrophoretic mobility (-,um.sec-' V-' .cm) and the length of the
`cell (jm) on 100 individual cells. Measurements were done 3 hr after irradiation with 50 kR. Closed
`circles are elongated cells with visible cleavage, which showed EPM above -1.2 jm-sec-'-V-'.cm
`as
`separated small cells and unirradiated cells.
`
`Page 6 of 11
`
`
`
`652
`
`SATO ET AL.
`
`E
`C 42D-
`
`D 3.0-
`E
`o
`
`@,
`
`0o 07O3
`
`D7
`
`OR
`
`1.0-
`0.0170033
`(17)
`(24)
`
`0.067
`0.100
`(12)
`(9.7)
`Ionic
`strength
`of
`ion
`(thickness
`atmosphere)
`FIG. 6. Relationship between the ionic strength of phosphate buffer used for the measurement of
`electrophoretic mobility of E. coli irradiated with 30 kR 30 min earlier (X) and of unirradiated E. coli
`(0). The vertical lines represent one standard deviation for 30 to 100 cells.
`
`0.134
`(8.4)
`
`0.167.
`(7.5)A
`
`various ionic strengths to determine whether a redistribution of charged groups in
`the membrane is responsible for EPM reduction. As indicated in Fig. 6, EPM
`decreased with increasing ionic strength. The difference in EPM between irradiated
`and nonirradiated cells was evident at ionic strengths of 0.100 and greater. At ionic
`strengths lower than 0.033, however, EPM was the same in irradiated and non-
`irradiated cells. Because the decay of potential with distance is less rapid in low-
`ionic-strength solution, the ionized groups embedded more deeply in the outer
`surface material exert their greater influence at lower ionic strength (1, 2, 12).
`Therefore with decreasing ionic strength, the thickness of the effective ionic layer
`increases. The thickness of the ionic layer was calculated according to the Debye-
`Hiickel equation by 3.06 X (ionic strength)-'/2 A as an approximate estimation
`assuming the complete dissociation of phosphates. These values are shown in pa-
`rentheses under each value of ionic strength in Fig. 6. The data can be explained
`by a vertical translocation of negatively charged groups from the outermost layer
`of 0-7.5 A into a deeper layer of 9.7-17 A, or the inverse translocation of positive
`charges from the deep layer to the outermost layer occurred in irradiated cells.
`Similar translocation of acidic sugars was suggested in our previous experiments
`using cultured mammalian cells and erythrocytes (11, 13).
`Effect of temperature and reagents on EPM change. Cells were irradiated with
`15 kR at 3?C on ice and then incubated in a water bath for 15 min with aeration
`
`Page 7 of 11
`
`
`
`SURFACE CHARGE AND CELL DIVISION
`
`653
`
`1.5-
`
`1.4-
`
`.:_4-
`
`OR
`
`o 1.3- ..
`
`.
`
`1.2-
`
`0
`
`LJ
`
`0.9-
`
`I
`
`,
`
`3
`
`'
`25
`15
`10
`35
`30
`20
`Incubation
`(C
`)
`temperature
`FIG. 7. Relationship between the incubation temperature and electrophoretic mobility (-,um-sec-'
`V-'.cm) 15 min after irradiation with 15 kR. The vertical lines represent one standard deviation for
`30 to 100 cells.
`
`at different temperatures. Figure 7 indicates that the decrease in EPM was max-
`imum and constant at temperatures ranging from 25 to 37?C, and smaller at
`temperatures of 10?C and lower. The slight difference of EPM between control
`and irradiated cells after incubation at the low temperature probably resulted from
`the EPM reduction during the measurement of EPM at 25?C for 5 min. The
`manifestation of the radiation effect on EPM depended markedly on temperature
`between 10 and 25?C. Incubation of unirradiated cells at different temperatures
`did not per se produce any effect on EPM.
`To estimate the factors involved in EPM reduction after irradiation, effects of
`agents which could modify the membrane were examined. Table I indicates that
`p-(chloromercuri)benzoic acid (PCMB, a sulfhydryl-blocking agent), fluorescein
`mercuric acetate (FMA, a sulfhydryl-linking agent), glycerin, and ethylene glycol
`bis(,/-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA, specific chelator of
`calcium ions) completely blocked EPM reduction after irradiation at the concen-
`trations at which the agent itself had no effect on EPM.
`
`DISCUSSION
`
`The present experiments demonstrate the simultaneous occurrence of reduction
`in EPM and inhibition of cell division shortly after irradiation and their concurrent
`recovery in individual cells of Escherichia coli. The advantage of cell electrophoresis
`was the simultaneous detection of EPM and cell morphology on individual cells
`seen directly under the phase microscope. Cells that were elongating due to the
`arrest of cell division after irradiation retained their reduced EPM. It should be
`noted that EPM of the cell is determined by the charge density per unit surface
`area independently of cell size or shape (3, 12). EPM reached the minimum 15
`min after irradiation, when the cells were only slightly longer than nonirradiated
`
`Page 8 of 11
`
`
`
`654
`
`SATO ET AL.
`
`TABLE 1
`
`Blocking of Mobility Change after X Irradiation by Modifying Agents
`
`Treatment
`
`No treatment
`30 kR, 30 min
`
`PCMB 5 X 10-6 M 30 min
`PCMB 5 X 10-6 M + 30 kR
`
`FMA 10-6 M
`FMA 10-6 M + 30 kR
`
`Glycerin 1 M
`Glycerin 1 M + 30 kR
`EGTA 1 mM
`EGTA 1 mM + 30 kR
`
`EGTA 1 mM + Ca2+ 1 mM
`EGTA 1 mM + Ca2+ 1 mM + 30 kR
`
`Electrophoretic
`mobility
`(m - sec-' V-' .cm)
`
`Significant
`difference
`(P < 0.05)
`
`-1.392 ? 0.076
`-1.011 ? 0.059
`
`-1.299 ? 0.062
`-1.252 ? 0.121
`
`-1.340 ? 0.074
`-1.331 ? 0.069
`
`-1.336 + 0.084
`-1.292 ? 0.054
`
`-1.322 ? 0.079
`-1.301 ? 0.086
`
`-1.329 ? 0.081
`-1.042 ? 0.067
`
`No
`
`es
`
`controls. Detailed analyses on alterations in cell number, cell length, and distri-
`bution of nuclear mass during the first 30 min of exposure were carried out. It was
`observed that the division of the cell proceeded if the daughter nuclei had already
`been segregated in the irradiated parental cell at the time of irradiation. Presumably
`septa had already been formed in these parental cells. Partition of new nuclei,
`however, was completely inhibited. After 30 min of incubation, enlargement of the
`condensed nuclear mass at the center of the cell and the elongation of the cell
`progressed without accompanying cell division but retained the reduced EPM. An
`important finding at the recovery phase was that not only the separated small cells
`but also long filamentous cells regained high EPM. The latter cells had a few
`cleavages at the cell termini, indicating the formation of dividing cross walls. This
`finding suggests that recovery of EPM in a whole elongated cell precedes or co-
`incides with the resumption of cell division at its termini. Since all small-sized cells,
`some of which lacked DNA, showed recovered EPM, cell division seemed to occur
`at the site where the surface structure had recovered irrespective of the distribution
`of DNA. This idea was supported by our recent experiment using a temperature-
`sensitive mutant which forms anucleated minicells at a high temperature (to be
`published).
`Negatively charged molecular species which are responsible for EPM in the
`bacteria have not been identified because of the absence of purified specific enzymes.
`It was shown that the anionic groups of the cell surface of E. coli begin to dissociate
`between pH 2 and 4 and are carboxylic in nature (14). A hypothetical mechanism
`which might lead to the reduction of EPM of E. coli after irradiation is considered
`to be a conformational change of membrane as suggested in the case of mammalian
`
`Page 9 of 11
`
`
`
`SURFACE CHARGE AND CELL DIVISION
`
`655
`
`cells (11, 13). Detection of reduced EPM only at low ionic strengths suggested a
`vertical translocation of charged molecules from the peripheral layer to a deeper
`layer after irradiation. The character of molecular conformational change was
`supported by the blocking of EPM loss by adding sulfhydryl-blocking agents or
`protein-linking agents or lowering the temperature of the cell culture. All these
`treatments seem to restrict the movement and rearrangement of membrane mol-
`ecules involving SH-proteins. The requirement of calcium ions for EPM reduction
`is another important feature of the mechanism. We reported the existence of a
`calcium-dependent process as an early step of EPM change after irradiation in
`erythrocytes (15). The target of X irradiation resulting in EPM reduction in mam-
`malian cells is thought to be the membrane itself, because EPM reduction occurred
`in the erythrocyte ghosts containing neither nuclei nor cytoplasm as well as in
`whole erythrocytes and cultured mammalian cells (11).
`Involvement of membrane damage in the loss of colony-forming ability of bacteria
`after irradiation has been proposed. A type of cell killing associated with the oxygen
`effect was suggested as the result of membrane damage (16, 17). Several mem-
`brane-specific drugs such as local anesthetics and tranquilizers preferentially sen-
`sitized the hypoxic cells to the same sensitivity as that of oxic cells (18). Further
`evidence in support of the hypothesis is as follows: (a) bacterial cells are sensitized
`by iodoacetamide under conditions that prevent the drug from entering the cells
`(18), (b) A mutants of E. coli carrying pol A- mutation and a mutant strain
`Bs- 1, both defective in a process of DNA repair, are not affected by the membrane-
`acting sensitizers (19-21). Because these membrane-acting reagents inhibit the
`repair of DNA (22), some interaction between the membrane and DNA was sug-
`gested in the process of sensitization. The DNA-membrane complex was indicated
`as a site which is particularly susceptible to enhancement of radiation damage by
`oxygen (23). Our results suggest that the inhibition and recovery of cell division
`are coordinated with the reduction and recovery of EPM, which reflects alterations
`in the structure of the cell surface after irradiation.
`RECEIVED: August 6, 1980; REVISED: November 12, 1980; RE-REVISED: February
`13, 1981
`
`REFERENCES
`
`1. D. H. HEARD and G. V. F. SEAMAN, The influence of pH and ionic strength on the electrokinetic
`stability of the human erythrocyte membrane. J. Gen. Physiol. 43, 635-654 (1960).
`2. A. H. MADDY, The chemical organization of the plasma membrane of animal cells. Int. Rev. Cytol.
`20, 1-65 (1966).
`3. J. N. MEHRISHI, Molecular aspects of the mammalian cell surface. In Progress in Biophysics and
`Molecular Biology (J. A. V. Butler and D. Noble, Eds.), Vol. 25, p. 1. Pergamon, Oxford/New
`York, 1972.
`4. M. H. REPACHOLI, Electrophoretic mobility of tumor cells exposed to ultrasound and ionizing
`radiation. Nature (London) 227, 166-167 (1970).
`5. C. SATO and K. KOJIMA, Change in electrophoretic mobility of cultured cells after X-irradiation
`and their modification by Sh-blocking agents and hemagglutinin. Radiat. Res. 60,506-515 (1974).
`6. H. RINK and H.-J. MEYER-TESCHENDORF,
`Influence of X irradiation on the electrophoretic mobility
`of yeast cells. Radiat. Res. 72, 317-324 (1977).
`7. C. SATO and K. KOJIMA, Irreversible loss of negative surface charge and loss of colony-forming
`ability in Burkitt lymphoma cells after X-irradiation. Exp. Cell Res. 69, 435-439 (1971).
`
`Page 10 of 11
`
`
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`656
`
`SATO ET AL.
`
`8. C. SATO, K. KOJIMA, M. ONOZAWA, and T. MATSUZAWA, Relationship between recovery of cell
`surface charge and colony-forming ability following radiation damage in three cell-lines. Int. J.
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