(CANCER RESEARCH 36, 3131-3137, September 1976]

SUMMARY

Cells of the rat basophilic leukemia cell line RBL-1 differentiate maximally when permitted to achieve growth arrestin a high-density stationary phase, in which the cell numberis constant, and the cells are arrested in a G phase of thecycle. Features of differentiation are the accumulation oflarge basophilic granules and increases in membrane meceptons for immunoglobulin E. However, changes in histamine content did not parallel granule development onchanges in immunoglobulin receptor concentration. Duringrapid “forcedexponential―growth, the cell number doublesevery 8 hr, 50% of the cells are in S phase, and diffenentiation is minimal.

INTRODUCTION

A transplantable basophilic leukemia, induced in a Wistarmatby administration of /3-chlonoethylamine p.o. (7), hasbeen adapted to tissue culture (19). Leukem mcbasophilsfrom both the tumor and the tissue culture-adapted line(RBL-1) have the characteristic features of differentiatedbasophils: prominent basophilic granules, substantial histamine levels, and large numbers of receptors for IgE (19).These unique cells have by now been used extensively byourselves and others. It seems appropriate, therefore, todescribe more completely the growth patterns of thesecells. We have found that the cells undergo striking vaniations in their degree of differentiation, as monitored bybasophilic granule development, and in their numbers ofreceptors for IgE. This paper describes the relationship ofmorphological differentiation to variations in cell cycle panametens. Changes in the receptors for IgE during the cellcycle have been described in detail elsewhere (17).

MATERIALS AND METHODS

Cell Culture. Cultures were grown as described previously (17) using 20% fetal calf serum. Tylocine, an antipleuropneumonia-like organism agent (Grand Island Biological Co., Grand Island, N. Y.), was added to culturemedia during the course of these experiments when Myco

, Presentaddress:Divisionof HematologyandOncology,DepartmentofMedicine, Childrens Hospital Medical Center, Boston, Mass. 02115.

2 To whom correspondence should be addressed.

Received July 30, 1975; accepted May 25, 1976.

plasma was cultured from other cell lines in the laboratory.No effect of Tylocine was noted on the growth of RBL-1cells. Cells were grown in 30- on 250-mI Falcon flasks on inspinner flasks.

Conventional Cytology. Cells were counted in a standardhemacytometer and viability determined by 0.08% trypanblue exclusion. Freshly prepared unfixed smears were prepared using a cytocentnifuge (Sakuna Fine Technical Co.,Tokyo, Japan) at 500 rpm and were stained with WrightGiemsa stain on an Ames Hematek slide stainer (Ames Co.,Division of Miles Laboratory, Inc., Elkhart, Ind.) by the Clinical Hematology Service, Clinical Center, NIH. Chromosomepreparations were made from exponential cultures after a30-mm exposure to Colcemid, 0.04 @g/ml(Grand IslandBiological Co.), using the method of Puck et a!. (25) andstainedas above.

Scanning Electron. Micrography. These studies werekindly performed by Dr. Raul Braylan, National Cancer Institute, Bethesda, Md. Washed cells (2 x 106/ml) were fixed incold 2.5% glutanaldehyde, pH 7.4, for at least 2 hr. Cells (1 x10w)were collected onto Flotnonic filter membranes (SelasFlotnonic, Spring House, Pa.) of 0.45-Mm pore size. Thefilters were rinsed, dehydrated in graded alcohols and unanyl acetate, and then, by the critical point method, coatedwith gold-palladium on a tilted rotatory stage, examinedwith an ETEC Autoscan at 20 kV and 45°tilt, and photographed with Polaroid 52 film.

Cell Cycle Determination. [3H]Thymidine, 0.36 Ci/mmole(Schwarz/Mann, Orangebung, N. V.), was added to culturesto a concentration of 1 mCi/mI. Pulse-labeled samples wereprocessed after 15 mm for determination of the S phase bythe “labelingindex―(22). Continuous [3H]thymidine labeling was used to determine the length of the G phase according to the technique of Maekawa and Tsuchiya (22).The percentage of labeled mitoses was used to determinethe length of the G2 phase (25). Slides were prepared forautoradiography by the technique used for chromosomeanalysis (25). They were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, N. Y.), dried, and stored at 4°.After 6 days of exposure they were developed in Kodak D-19developer, fixed, and stained @nthe Ames slide stainer.Nuclei with more than 10 overlying grains (x3 background)were scored as labeled.

Automated Cytology. Cell counts and volume distributioncurves were obtained using a Model B Coulter counterequipped with a Coulten channelizer and an X, Y plotter(Coulter Electronics, Hialeah, Fla.). Analysis of relative sin

SEPTEMBER1976 3131

Cell Cycle and Morphological Changes during Growth andDifferentiation of a Rat Basophilic Leukemia Cell Line

Donald N. Buell,1 B. J. Fowlkes, Henry Metzger, and Chaviva lsersky2

Immunology Branch fD. N. B.) and Laboratory of Pathology (B. J. F.J, National Cancer Institute, and Section on Chemical Immunology, Arthritis andRheumatism Branch, National Institute of Arthritis, Metabolism and Digestive Diseases (H. M. , C. I.J, NIH, Bethesda, Maryland 20014

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D. N. Buelleta!.

tube down through the gradient. A polyethylene tube wasattached to the stainless steel tube, and the fractions werecollected by siphoning. The cells in the gradient fractionswere counted, their volume distribution was determined,and the distribution of DNA content was assessed by FMFanalysis.

RESULTS

General Characteristics of Cell Line. In stationary flasksRBL-1 cells generally form a sparse monolayer with overlying cells in suspension proliferating to high densities. Nosuch attachment occurs in spinner flasks with the cells keptin continuous motion, and the cells reach densities of 2 x106 cells/mI or higher. Reproducible growth patterns, highviabilities in prolonged stationary growth, and high levels ofreceptors for IgE were found using medium supplementedwith 20% fetal calf serum.

During routine maintenance feeding schedules, RBL-1cultures undergo a pattern of a lag (12 to 36 hr), 1 to 3doublings, and then a stationary phase where they can bemaintained for 48 to 96 hr with high viability (>90%). Whenstationary-phase cells are centrifuged and resuspended infresh medium at 3 x 10@cells/mI, they undergo a lag phaseduring which the cells increase in volume and pass throughthe G1, 5, and G2 phases of the cell cycle in partial synchrony as indicated by FMF analysis of their DNA content(Chart 1). After the exponential phase the cells enter aprolonged stationary phase with a decreasing average cellvolume. Such cells have the G content of DNA (Chart 1).

RBL-1 cells could be kept in “forcedexponential―growthby removing two-thirds to three-fourths of the cell suspension daily and restoring the volume in the spinner flask withfresh medium. When cells that had been in rapid exponential growth for a few generations were suspended in freshmedium at 3 x 10@cells/mI without further feeding, thecharacteristic pattern of growth into a high-density stationary phase occurred. However, after 8 to 10 days of forcedexponential growth, such cells lose the capacity to achievea high-viability prolonged stationary phase. The cells growquickly to high densities and die, apparently before undergoing the metabolic alterations necessary for prolongedstationary phase survival.

Lag-phase cells enter S phase between 9 and 16 hr afterresuspension in fresh medium. In other cultures the lengthof time prior to the start of S phase varied, being longer forcultures with longer stationary phases. Similarly, the totalduration of the lag phase (range, 16 to 36 hr) appeared tocorrelate with the length of time during which the cells werein stationary phase. We have not determined whether thelength of time in S phase is also variable and contributes tothe variation in the duration of the lag phase on whether themain variability is in the time that elapses before the ‘‘resting―cellscan reenterthe growth cycle.

Cell Cycle Parameters. Using [3H]thymidmneincubationand autonadiognaphy, the durations of the cell cycle and itsG1,5, and G2phases were determined for a rapidly growing“forcedexponential―culture with a doubling time of 8 hr. A15-mm pulse of [3H]thymidine labeled 50% of cells (labelingindex, 0.5). Since the cell cycle time, T(., = 8 hr, the duration

gle-cell DNA content was performed by FMF2 using themithnamycin method on a Los Alamos Scientific Laboratorymultiparameter cell sorter (5, 6, 31). For the latter studiescells were centrifuged at 1000 rpm, washed with filtered0.85% NaCI solution , fixed in 75% ethanol containing 15 mMMgCl2, and stored at 4°until analyzed. For the FMF analysis,2 x 10@cells were suspended in 4 ml of 0.15 M NaCl-0.01 MNaPO4,pH 7.4, containing mithnamycin, 100 @g/ml.After 20mm at room temperature,samples were nun on the cellsorter, and the oscilloscope pattern of single-cell DNAquantitations was recorded on Polaroid type 105 positivenegative film (Polaroid Corp. , Cambridge, Mass.).

The FMF patterns were converted to digital form by projecting the polaroid transparencies on graph paper using anenlarger at a fixed setting. Relative numbers of cells contaming G1,5, and G2quantities of DNA were estimated fromthe distributions using the Simulation, Modeling and Analysis 26 computer program (2, 3) with constraints similar tothose used by Dean and Jett (6). However, the inter-G1-G2(S-phase) distribution curve was not constrained to fit a2nd-order polynomial but instead was assumed to resultfrom a series of 10 gaussian distributions with means andspreads evenly spaced between the extremes determinedfor the G, and G2 populations.

Thymidine Block and Synchronization. To achievegrowth arrest in S phase (13), thymidine was added to aculture to give a final concentration of 2.5 mM. Synchronization by double thymidine block was accomplished by amodification of the technique of Galavazi et a!. (13). Cells inculture were exposed to 2.5 mM thymidine for 8 hr, centnifuged for 10 mm at 130 x g at room temperature, washed inmedium with 10@ M deoxycytidine, (Schwarz/Mann), mecentrifuged, and suspended in medium without nucleotides. After 12 hr 2.5 mM thymidine was again added for 8hn, and the cells were centrifuged, washed in fresh medium,centrifuged, and resuspended in medium with 10_6M deoxycytidine.

Separation of Cells by Velocity Sedimentation on FicollGradients. The technique described for human lymphoidcells (9) was modified for RBL-1 cells. Gradients (5 to 10%,w/v) were prepared by dissolving Ficoll (Pharmacia FineChemicals, Piscataway, N. J.) directly in bicarbonate-freeEagle's Spinner No. 1 medium containing 0.01 M N-2-hydroxyethylpiperazine-N ‘-2-éthanesulfonicacid buffer, penicillin, streptomycin, Tylocine, and glutamine but no serum.The solutions were sterilized by filtration through a Nalgenefilter unit with a 0.45-.@m membrane (Nalge Sybron Co.,Rochester, N. V.). A 5 to 10% linear continuous gradientwas prepared using a Buchlen Instrument density gradientgenerator. The final gradient was 80 ml in a 3- x 10.5-cmcylindrical No . 32086 polycarbonate tube (InternationalEquipment Co. , Needham Heights, Mass.). An additional 5ml of a 5% Ficoll solution were layered on top of the gradient. A monodisperse cell suspension in medium containing 2 to 3 x 10@cells at 5 x 106cells/mI was layered on topof the gradient. The samples were centrifuged for 30 mm at80 x g at room temperature in an International PR-2 centnifuge using a swing-out rotor. Fractions of 4 ml each werecollectedby carefullyplacinga 0.2-x 15-cm stainlesssteel

2 The abbreviation used is: FMF, flow microfluorometry.

CANCER RESEARCH VOL. 363132

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@jr

TI@L:

Time (hr)Cells/mI(x

10')Labelingin

dex[3H]Thymidine

incorporation―(cpm x10@)07.727.70.8212.347.80.8116.877.90.355.21410.54.92415.80.255.23219.90.09―3.3―

Basophi!ic Leukemia Cell Changes

1) indicated that the cells were in S phase 2 and 4 hr afterrelease from thymidine block and then passed through G.,before mitosis. In a 2nd double thymidine-synchronizedculture, FMF analyses of single-cell DNA content distnibutions were included. At 10 hn, just after the 1st synchronousdivision and cell doubling, all cells had the G content ofDNA. Subsequently, the cells passed parasynchnonouslythrough S and G2as indicated by the DNA patterns.

Gradient Fractionation by Rate Zonal Centrifugation.Three different cultures, a forced exponential culture andcultures analyzed at the late exponential and early stationary stages, were analyzed. The cell distribution profilesdiffered for cells from different stages of growth with thelarger cells of the “forcedexponential' ‘culture penetratingdeepen into the gradient than those of a transitional orstationary culture. Chart 2 presents analyses of culturessampled in late exponential and early stationary stages ofgrowth. Chart 2a shows the Coulter counter volume distnibutions for the 2 cultures. Each of these shows a considenable spread of cell volumes within the parent cultures. Chant2b showsthat the individual gradient fractions show a muchnarrower distribution of cell volumes and that the depth ofpenetration into the gradient correlates directly with the cellvolume. Chant 2, c and d, presents the DNA distributionpattern of the parent cultures and of the derived fractions.Analysis of the distribution of cells in the different cell cyclephases derived from the FMF data showed that separationby size achieves separation by cell cycle parameters, withthe larger G2-phase cells enriched in the bottom fractions,G cells predominating at the top, and S-phase cells enniched in the intermediate fractions (Table 2). Although theanalysis of the parent culture in the early stationary phaseshowed no significant numbers of G2 cells, the gradientfractionation demonstrated their presence. The G cell contamination in the lower fractions probably results from“trailing―due to the large excess of G cells in the parentculture, G cells that sedimented as doublets, M cells completing mitosis during on after centnifugation (at room temperature), large G cells ready to enter S phase, or cellscarried down by passing the sampling tube through thegradient.

Morphological Differentiation. RBL-1 cells vary in appearance from primitive cells resembling pnomyelocytes to

Table 1

Ohr

ci:w

::iz-J-Jw0

w>

I—

-JLiJci:

CHANNEL NUMBER(Relative DNA content)

Chart 1. FMF analysis of DNA content in cells from a culture during a lagphase of growth. At 0 time the cells had just been diluted to 3 X 10' cells/ml after 3 days of stationary growth at 2 x 10@cells/mi. No change in cellnumber was observed till 31 hr, at which time the cells numbered —4x 10'cells/mI.

of S phase,@ —4 hr (from labeling index = Js/J(). Labeledmitoses were absent after 1 hr of exposure to [3Hjthymidmne,but 95% of mitoses were labeled by 2 hr; thus, the durationof G,@ = —1.5 hr. The length of G was determineddirectly by following the disappearance of unlabeled cellsfrom the culture during continuous exposure to[3H]thymidine (22). After 1 to 2 hr no unlabeled cells enteredmitosis. The decrease in unlabeled cells after that timeserves as a measure of the passage of cells from G into Sphase. Between 1 and 4 hn, the percentage of unlabeledcells fell to 0. Thus G was 2 to 3 hr long. The rate of entry ofcells into S (—50%in 4 hr) was consistent with an 8-hr cellcycle time. The viability of the forced exponential cultureswas 100@/@and all cells were cycling rapidly, as indicated by100% labeling of cells with [3H]thymidmnein 4 hr.

Cell Cycle Synchronization Studies. Cultures that wereexposed to excess thymidine for prolonged periods of timeshowed a state of ‘‘unbalanced growth' ‘in which the cellsgrow but do not synthesize DNA or proliferate (21, 29). FMFanalysis confirmed that the cells were still in early S phaseeven after25 hr.Cellsexposed to excess thymidine twicefor 8 hr with an interval of 12 hr were similarly arrested butresumed growth upon elimination of the thymidine andaddition of deoxycytidmne. [3H}Thymidine incorporation mesuIts and [3H]thymidine pulse autoradiography data (Table

Cell count and DNA synthesis after release from double thymidineblock

‘1Counts incorporated by 1 x 10@ cells/mI incubated for 15 mm

with 1 mCi of [3H]thymidmne per ml, spun down, washed with 0.15 MNaCI-0.01MNaPO4(4°),dissolvedin 1 ml Hyamine,andcounted.

1@Cultures approaching stationary-phase conditions.

SEPTEMBER 1976 3133

GI G2

(a) H@1

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Analysisof cells from early stationary culture fractionated onaFicollgradientFMF

analysis(%)Fraction

Cells/mi (x 10 ‘) G1 S G2 +MTotal

69 29280.84 18 483491.30 29 656101.91 72 280112.90 92 80121.90 99 10130.87 100 00Table

3Histamine

content of cultured RBL-1cellsHistamineCells/mI

(ng/106Culturestage Time (hr) (x l0@)cells)Early

stationary 72 2.250024-hrstationary96 2.146048-hr

stationary 120 2.147096-hrstationary 144(0)― 2.2 (0.3)―300Lag

148(4) 0.3430Lag156(12) 0.3530Start

exponential 162(24) 0.33600Midexponential192(48) 1.0670Forced

exponential 120 0.95500Forcedexponential144 1.0690Forced

exponential 216 0.8730‘IAfter dilution with fresh medium.

D. N. Buell et al.

d copy by Dr. K. Becker. The photographs were in addition

kindly reviewed with us by Dr. R. Hastie. The cells showmany of the features seen in normal human basophils (15)with no unusual features; no viral-like bodies were observed. The cells have also been examined by scanningelectron microscopy (Fig. 2c). The predominant topologyconsists of hundreds of villi, fine veil-like surface protnusions being a less common feature. Cells from a “forced

_______exponential―andastationary-phasecultureappearedsimilamby scanning electron microscopy.

Chromosome counts were performed on 20 cells. Fivegave a tetraploid value of 84 while the remainder werehypotetnaploid, 1 cell having as few as 70 recognizable

_______chromosomes.Despitetheheteroploidy,single-cellDNAquantitation using FMF analysis revealed characteristic pattemns representing the G, 5, and G2 populations so thatthese cells appear to have a stable DNA content (18). Nofurther kanyotyping has been performed.

Histamine Content. A limited number of cultures werefollowed for histamine content during culture growth usingan automated procedure (28). The results of 1 such expeniment are given in Table 3. It can be seen that in exponentialgrowth there was no decrease in histamine content per ce!!despite the rapid divisions; if anything there was a smallincrease. Several other cultures showed qualitatively similarresults except that fairly substantial decreases in histaminecontent were usually seen in the latter part of the exponential phase.

We never observed a substantial increase in histamine inthe stationary phase of culture growth comparable to the

Table 2

G1G2

:3f.Al2J@,

ill

:ok-

8@

i7@

CHSNN[LNUMBERRELATIVEDNACONTENT

a

z

b

:3z

Chart2. Left: volumedistribution spectraof cells from late exponential(ExpO) and early stationary (Stat) cultures (a) and in Ficoll gradient fractionsof these 2 cultures (b). Numbers over peaks, fraction numbers. All ordinatescales are linear. Right: c, distribution of cells in G, 5, and G, + M phases ofthe cycle in a late exponential culture and in Ficoll Gradient Fractions 6 to 12from it; d, the distribution in an early stationary culture and in Ficoll GradientFractions 7 to 13 from it. The ordinate and abscissa scales are linear.

well-differentiated basophils, depending on growth conditions (Figs. 1 and 2). The content and size of basophilicgranules in RBL-1 cells varied depending on the state ofgrowth of the culture. Cells from “forcedexponential―cultures uniformly had only a few small granules (Fig. la). Theappearance on a Wnight-Giemsa-stained smear was that ofprimitive cells of myeloid origin (promyelocyte) with small,sparse granules, so that the cells could not be easily identified as basophils. Approximately 50% of the cells in suchcultures are passing through the G phase of the cell cycle.As the cultures entered and remained in stationary phase,basophilic granules increased in number and became moreprominent. In late stationary phase, these cells uniformlyhad the appearance of well-differentiated basophils withlarge granules (Fig. lb). As has been shown, these cellshave a G1DNA content.

When stationary-phase cells were resuspended in freshmedium at lower density (lag phase), the basophilic granules remained intact until the 1st cell division (Fig. 2a).Thereafter, granules decreased in size and number as thecells passed through successive divisions (Fig. 2b). Duringroutine maintenance, with twice-weekly feeding of the cultune, the cells remained fairly well differentiated, with prominentgranules.

Granule development was dependent upon growth of theculture into high-density stationary phase. When growth ofan exponential culture was arrested with excess thymidine,granule development was that characteristic of the startingexponential culture, even after prolonged thymidine arrest.

Shortly after we received the tumor, some thin sections ofcells from tumor minces were examined by electron micros

3134 CANCER RESEARCH VOL. 36

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Basophi!ic Leukemia Cell Changes

can be considered to be arrested in the G phase on to beresting “G0―cells (8, 10, 20). However, G cells in theseinstances may have markedly different properties than dothe G-phase cells of a cycling, exponentially growing cultune (1). This is clear from our results where G cells from anexponential culture have markedly decreased basophilicgranules and IgE receptors (17) compared with G cells ofsimilar size from stationary-phase cultures.

The nature of the transition from stationary phase toreentry into the cell cycle should be examined further. Duning the usual 20- to 24-hr lag in growth, up to 16 hr mayelapse before the cells enter the S phase. This may be thetime necessary for the cells to revert from a G, or “stationany-phase G―and to reenter the G phase of an active cycle(1, 8, 10). A block in progression to terminal differentiation,the state where the cells are unable to reenter the cycle,appears to be a feature of leukemias (14) but is not absolute.Friend virus-induced munine emythnoleukemias (12) andgranulocytic leukemias of mouse (11) and man (23) can beinduced to undergo terminal differentiation in vitro. Additional data would be required to determine whether RBL-1cells in prolonged stationary-phase culture even reach thestate of terminal differentiation.

The differentiation of normal basophils has been studiedin guinea pig bone marrow during a serum-induced basophil response (30). Studies in other species have been lessextensive because of the relative paucity of basophils innormal marrow (32). These electron microscopic studieswere based on identification of cells as basophils by granuleappearance and correlations with the degree of nuclearmaturation. Rat mast cell differentiation has also been studied (4, 24), but again the order of events had to be inferredand could not be directly followed. Precise time sequencestudies of basophil differentiation are impossible usingsuch techniques. To the extent that the RBL-1 leukemicbasophil cultures mirror normal basophil (?mast cell) differentiation, detailed studies should now be possible.

ACKNOWLEDGMENTS

We are grateful to Dr. F. K. Millar (Laboratory of Theoretical Biology,National Cancer Institute) for developing the computer program used toanalyze the FMF data, to Dr. R. Siraganian (Laboratory of Microbiology andImmunology. National Institute of Dental Research) for performing the histamine analyses, to Dr. Raul Braylan for the scanning electron micrography,and to M. Cassidy and Dr. C. Herman, Cytology Automation Program (Laboratory of Pathology, National Cancer Institute) for their cooperation andassistance.

REFERENCES

1. Backer, H. , Stanners, C. P., and Kudlow, J. E. Control of Macromolecular Synthesis in Proliferating and Resting Syrian Hamster Cells in Monolayer Culture. II. Ribosomal Complement in Resting and Early G, Cells. J.Cellular Physiol.. 77: 43-50, 1971.

2. Berman, M., Shan, E., and Weiss, M. F. The Routine Fitting of KineticData to Models: A Mathematical Formalism for Digital Computers. Biophys. J.. 2: 275-287, 1962.

3. Berman, M. , and Weiss, M. F. Simulation, Analysis and Modeling Manual. U. S. Public Health Service Publication No. 1703. Washington, D. C.:U. 5. Government Printing Office, 1967.

4. Coombs, J. W., Lagunoff, D., and Benditt, E. P. Differentiation andProliferation of Embryonic Mast Cells of the Rat. J. Cell Biol. , 25: 577-592, 1965.

5. Crissman, H. A., and Tobey, R. A. Cell Cycle Analysis in 20 Minutes.Science, 184: 1297-1298, 1974.

observed morphological changes on increases in receptorsfor IgE (see below). The histamine values shown in Table 2are similar to those most frequently observed oven thecourse of the 1st 1.5 years that the cells were cultured.Recently, our cultured cells have shown substantially lowerhistamine contents.

DISCUSSION

Leukemic basophils of the RBL-1 cell line can be inducedto undergo reproducible patterns of growth and diffenentiation . The length of the G , 5, and G, phases of the cell cyclewas determined only for the “forcedexponential―culture.Under these conditions, all the cells cycle rapidly and thephases have minimal variation (26). Under any other conditions, determination of the lengths of G, S and G2 phaseshas little meaning with changes in cycle parameters takingplace more rapidly than they can be studied by such techniques as percentage-labeled mitosis curves (26, 27).

The growth patterns have been correlated with variationsin cell cycle parameters. Stationary-phase cells are small,are arrested in the G phase of the cell cycle, and undergodifferentiation as manifested by large prominent basophilicgranules and 3- to 5-foldincreases in the IgE receptorconcentration (17). By contrast, cultures kept in rapid‘‘forcedexponential― growth have minimal differentiationwith fewer, smaller granules and lower levels of IgE neceptons. Paradoxically, the histamine content, which might beconsidered a differentiated characteristic of the cells, isquite low in the stationary-phase cultures. We are uncertainas to the cause of this apparent discrepancy. Stationarycells are more fragile. It is more difficult to make good cellsmears from such cultures, and they show an increasedleakage of histamine when incubated in test tubes. This maypartially explain the lower values observed for the stationarycells. Attention should also be called to the fact that thehistamine content has varied greatly. Although many of thecultures have values such as 400 to 600 ng/106 cells, occasionally values as high as 1050 to 1400 ng/106 cells wereseen; however, values as low as 50 to 150 have been seenmore recently. We are unable to explain satisfactorily theseoscillations.

In 5- to 6-day-old Sarcoma 180 cultures, characterized byhigh density and decreasing growth rates, traditional techniques of autoradiography following pulse [3H]thymidinelabeling (27) give varying results, depending on such factons as duration of emulsion exposure and grain countthreshold. In those studies, correlations of cycle parametersdetermined using [3H]thymidine incorporation-autoradiography with cell cycle determinations based on single-cellDNA quantitation by FMF analysis revealed low levels ofDNA synthesis in cells with the G, content of DNA (27). Insuch cultures, S phase does not appear to exist as traditionally defined (16); low levels of DNA synthesis may occurthroughout most of the cell cycle. A similar situation mayexist in RBL-1 cultures in stationary phase, where all thecells appeared to be in G by FMF analysis but low levels of[3H]thymidine incorporation were still measurable (17). Inthese RBL-1 stationary-phase cultures, as mother culturesthat manifest high-density growth inhibition (16), the cells

3135SEPTEMBER1976

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D. N. Bue!l et a!.

6. Dean, P. R. , and Jett, J. H. Mathematical Analysis of DNA DistributionsDerived from Flow Microfluorometry. J. Cell Biol., 60: 523-527, 1974.

7. Eccleston, E., Leonard, B. J., Lowe. J. S., and Welford, H. J. BasophilicLeukemia in the Albino Rat and a Demonstration of the Basopoietin.Nature New Biol. , 244: 73-76, 1973.

8. Epifonova, 0. I. , and Terskilsh, V. v. On the Resting Periods in the CellLife Cycle. Cell Tissue Kinet., 2: 75-93, 1969.

9. Everson. L. K., Buell, D. N., and Rogentine, G. N. , Jr. Separation ofHuman Lymphoid Cells into G,, 5, and G2Cell Cycle Populations by avelocitySedimentationTechnique.J. Exptl.Med., 137:343-358,1973.

10. Fahey, J. L., Buell, D. N., and Sox, H. C. Proliferation and Differentiationof Lymphoid Cells: Studies with Human Lymphoid Cell Lines and Immunoglobulin Synthesis. Ann. N. Y. Acad. Sci., 190: 221-234, 1971.

11. Fibach, E., Landaw, T. , and Sachs, L. Normal Differentiation of MycloidLeukemic Cells Induced by a Differentiation-Inducing Protein. NatureNew Biol., 237: 276-278, 1972.

12. Friend, C., Scher, W., Holland, J. G., and Sato, T. Hemoglobulin Synthesis in Murine Virus Induced Leukemic Cells in vitro: Stimulation ofErythroid Differentiation by Dimethyl Sulfoxide. Proc. NatI. Acad. Sci. U.S.,68:378-382,1971.

13. Galavazi, G. . Schenk, H. , and Bootsma, D. Synchronization of Mammahan Cells in vitro by Inhibition of the DNA Synthesis. I. Optimal Conditions. Exptl. Cell Res., 41: 428-437, 1966.

14. Gavasto, F. Granulopoicsis and Cell Kinetics in Chronic Myeloid Leukemia. Cell Tissue Kinet., 7: 151-163, 1974.

15. Hastie, R. A Study of the Ultrastructure of Human Basophil Leukocytes.Lab. Invest., 31: 223—231,1974.

16. Howard, A., and PeIc, S. R. Synthesis of Deoxyribonucleic Acid inNormal and Irradiated Cells and Its Relation to Chromosome Breakage.Heredity, 6(Suppl): 261-273, 1952.

17. Isersky, C., Metzger, H., and Buell, D. N. Cell Cycle Associated Changesin IgE Receptor during Growth and Differentiation of a Rat BasophilicLeukemia Cell Line. J. Exptl. Mcd., 141: 1147-1162, 1975.

18. Kraemer, P. M., Deaven, L. L., Crissman, H. A., and Van Dilla, M. A. DNAConstancy Despite Variability in Chromosome Number. Advan. Cell Mol.Biol.2:47-108,1972.

19. Kulczycki, A., Jr., lsersky, C., and Metzger, H. The Interaction of IgE RatBasophilic Leukemia Cells. I. Evidence for Specific Binding of IgE. J.Exptl. Med., 139: 600—616,1974.

20. Lala, P. K. , and Paft, H. M. A Characterization of the Boundary between

the Cycling and Resting States in Ascites Tumor Cells. Cell Tissue Kinet.,1: 137-146, 1968.

21. Lambert, W. C., and Studzinski, G. P. Thymidine as a SynchronizingAgent. II. Partial Recovery of HeLa Cells from Unbalanced Growth. J.Cellular Physiol. , 73: 261-266, 1969.

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3136 CANCER RESEARCH VOL. 36

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Basophi!ic Leukemia Ce!! Changes

la

2a 2b

SEPTEMBER 1976 3137

‘41

Fig. 1. a, cellsfroma “forcedexponential―culture:b, cellsfroma late-stationary-phaseculture.Wright-Giemsa-stainedcytocentrifugepreparations.Finalmagnification. x 630.

Fig. 2. a, cellsafter 21 hr in lag phaseof growth:b. cells from sameculture as in a after -13 hr of exponentialgrowth (37hr after resuspension)a, b,Wright-Giemsa-stained cytocentrifuge preparations photographed at x 320 and magnified x 2.5 from negatives); c, scanning electron micrograph of cellsfrom a stationary culture. Final magnification, x 5300.

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1976;36:3131-3137. Cancer Res   Donald N. Buell, B. J. Fowlkes, Henry Metzger, et al.   Differentiation of a Rat Basophilic Leukemia Cell LineCell Cycle and Morphological Changes during Growth and

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