Quantitative Studies on Mammalian Cells in Vitro

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  • REVIEWS OF MODERN PHYSICS VOLUME 31, NUMBER 2 APRIL, 19S9

    II l g~T. T. PUCK

    Department of Biophysics, University of Colorado Medical Center, Denver 20, Colorado

    ~ 'HE major advances of the science of genetics havecome about through the use of two fundamental

    types of operations. The first and oldest consists inmating of selected, multicellular plants or animals toproduce large numbers of offspring, among which thedistribution of various inherited characters is examined.This procedure has produced that body of knowledgeknown as classical genetics and which, beginningsystematically with the studies of Mendel, has deline-ated the processes which sustain and modulate bio-logical heredity in all living forms. This experimentalapproach, which essentially examines genetic mecha-nisms in the germ cells, has been extremely successfulin elucidating hereditary phenomena in organisms asdiverse as Drosophila and Zea mays, but is difhcult toapply to the slowly and less extensively reproducingorganisms like the mammals. Thus, while some notableadvances have been accomplished, it has not beengenerally possible to obtain suKciently large numbersof progeny from selected mammalian matings to providepopulations adequate for measurement of many im-portant genetic events.

    The second major class of genetic operations involvesa more recent technique, which consists in study of theindependent micro- and ultramicro-organisms thefree-living cells and the viruses. Here the standard unitis a single cell or particle, and the fundamental operationinvolves examination of the distribution of genetictraits among the progeny of asexual reproduction. Theprincipal power of this method lies in the vast multipli-cation factor which it aBords. Instead of hundreds orthousands, millions or billions of progeny, all arisingfrom a single individual, can be rapidly produced andquickly scanned for a large variety of genetic characters,so that even rare events can be quantitatively examined.In the last twenty years, such genetic studies onmicroorganisms have produced a whole new field ofknowledge. Sexual methods of genetic analysis are notexcluded from these operations but can sometimes bearranged in a step preceding the clonal multiplicationof the single cells. In addition, however, new methodsfor nonsexual genetic analysis have been found whichpromise to be exceedingly productive. Among thefundamental results which have issued from use ofsingle-cell techniques applied to microorganisms duringthe last two decades are included: (a) the first system-atic demonstration of the direct relationship betweengenes and enzymes; (b) delineation of many specific,metabolic pathways involved in gene-controlled bio-

    ~ Contribution No. 79 from the Florence R. Sabin Laboratories.

    syntheses; (c) demonstration of the direct exchangeof genetic materials between cells previously consideredto multiply only mitotically; (d) introduction of genesinto cells by means of temperate viruses or puredeoxyribonucleic acid; (e) the most accurate measure-ment of gene-mutation rates; (f) demonstration andquantitative analysis of the processes involved in en-zyme induction among genetically competent cells inresponse to specific environmental stimuli; (g) delinea-tion of the characteristics of linear inheritance in bac-teria; (h) systematic use of mitotic crossing-over incertain organisms to localize genes on their chromo-somes; and (i) the most detailed mapping at the levelof molecular dimensions of the linear sequence of geneticdeterminants, as accomplished in a bacteriophagechromosome.

    An index of the fruitfulness of this latter approach tothe analysis of genetics and related metabolic processesis afI'orded by the fact that the great majority of thediscussion devoted to genetic processes in this bio-physical conference has been concerned with develop-ments arising directly from microbial genetics.

    In our laboratory, a program was initiated, attempt-ing to develop a methodology that would make possiblestudies of mammalian cells by means of the techniquesof microbial genetics. If successful, such a methodologywould provide a new avenue for exploration of mam-malian genetic processes among the somatic cells ofmulticellular organisms, which would complementstudies on the genetics of the germ cells achieved byconventional mating studies. In addition, it wouldaGord all of the many kinds of analyses which an un-limited number of progeny provides. Such techniquesmight facilitate elucidation of the nature of any changesin the genome and phenome, respectively, which areresponsible for the characteristic behavior of the variousdifI'erentiated tissue cells; localization of diferent geneson the various chromosomes; provision of mutated cellscontaining specific biochemical blocks for the deline-ation of various enzymatic steps through quantitativestudy of specific biosyntheses, like that of hormones andantibodies in specialized cells; search for processes likesexual genetic exchange and transduction in somaticcells, and investigation of the genetic biochemistry ofnormal and malignant cells.

    Participating in this program have been a series ofdevoted co-workers: Steven Cieciura, Harold Fisher,and Philip Marcus carried out studies which formed thebasis of their doctoral theses; and D. Morkovin, G.Sato, and N. C. Webb joined this program in a post-doctoral capacity. Chromosome studies have been

    433

  • 434 T. T. PUCK

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    FzG. 1. (a) Petri dish which was seeded with 100 single cells ofthe S3 strain of the HeLa culture, an aneuploid which originatedin a human cancer. (b) Petri dish seeded with single cells of aeuploid human strain. These cells tend more to spread and migrate,and so the colonies tend to run together unless a smaller numberof cells is plated or a larger plate is employed.

    carried out initially in a collaborative arrangement withDr. Chu and Dr. Giles at Yale University, and morerecently with J. H. Tjio, who joined our laboratory lastyear. Dr. Arthur Robinson has conducted those aspectsof these studies which involved work on human patients.

    The first step in this program, which, of course, hasdrawn a great deal on previously existing tissue-culturemethodology, was to develop means for plating singlemammalian cells, under conditions such that everysingle cell develops into a discrete, macroscopic colony. 'This is essential in order to quantitate growth of singlecells of a large population, and to make possible isola-tion of mutant clones. A simple, quantitative means forgrowth of single cells into colonies was achieved by a

    procedure, identical in principle to, and only slightlymore complex in practice than, the plating methodwhich constitutes the basis of quantitative microbiology.Figure 1(a) illustrates a representative plating in which100 cells of the S3 HeLa clone, plated in a Petri dishin nutrient medium, have attached to the glass andproduced, within the limits of sampling uncertainty, adiscrete macroscopic colony from each cell inoculated.The counting of such colonies is completely objectiveand highly reliable, so that this methodology permits allof the types of experiments characteristic of quantita-tive bacteriology to be applied to mammalian cells.Cells taken from organs as diverse as skin, liver, spleen,bone marrow, testis, ovary, kidney, lung, and others,from man and other mammals, and from individuals ofa large variety of ages, all respond similarly. It can beconcluded that at least large numbers of cells exist invirtually every organ of the mammalian body whichretain the potentiality of growth as independent micro-organisms, if provided with an adequate physical andchemical environment.

    By and large, two general types of cellular andcolonial morphologies result from such plating. Thatillustrated in Fig. 1(a) has been called "epithelial-like"because the cells form compact, tight colonies withrelatively smooth, well-defined edges. In our experienceso far, the cells that tend to grow stably in this fashionhave been polyploid, and. usually aneuploid. The othertype of colony, which has been called "fibroblast-like"but should perhaps be referred to simply as "stretched, "is illustrated in Fig. 1(b), which shows the tendency ofthe colonies to grow with rough edges, and of the cellsto line up as elongated parallel structures. This stretched,needle-like conformation is more characteristic of theeuploid cells grown by the methods developed in our lab-oratory. However, the molecular environment stronglyinfluences the morphology of cells grown in this manner. '

    The course of developments in microbial geneticshas demonstrated the enormous utility of quantitativesingle-cell plating for the isolation of stocks with raregenetic markers that permit analytical experimentation.Unless each cell can grow in isolation to form a colony,it becomes impossible to apply the highly discriminatingscreening methods by which millions of cells are sub-jected to a stressful situation permitting growth of onlyoccasional rare mutants, for otherwise, the exceedinglyrare mutant which though potentially is capable ofreproduction is not part of a large reproducing popu-lation and may not be enabled to express its growthpotentiality. An effective single-cell plating techniquepermits ready recognition of even very rare geneticevents, and quantitation of their frequency.

    Isolation of mutant clones from such plates is astraightforward process for which a variety of diferentmechanical methods have been developed. We haveestablished mutant clones which have arisen spontane-ously or have been induced as a result of x-irradiation.Among these are included forms with divergent nutri-

  • STUD I ES ON MAM MAL IAN CELLS IN VITRO 435

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    Fro. 2. Demonstration of some representative mutant strainsisolated from the HeLa population. (a) Plating efficiency curvesin the presence of diferent amounts of human serum of 2 mutantsfound to occur spontaneously in the original HeLa population.The cellular and colonial morphologies of these two strains areidentical but their growth requirements are diferent. (b) Distribu-tion of chromosome number in 2 HeLa clones. The clone at thetop (S/NDV) has the same chromosome number as S3, but ischaracterized by its resistance to destruction by Newcastle diseasevirus.

    tional requirements for colony formation, changedcolonial morphologies, resistance to destruction byspecific viruses like Newcastle disease virus, and di6er-ences in chromosomal constitutions (Fig. 2). Thesemutants have been grown for months or years incontinuous culture, during which they have producedastronomical numbers of progeny, and have exhibitedstability with respect to their identifying characteristicswhich is in every way comparable to that of thefamiliar mutants in bacteria and molds. "

    Attention was next devoted to the development of adefined chemical medium which would promote growthin high efficiency of single mammalian cells, and so

    make available means for isolating mutants with specificnutritional markers. Many laboratories have contrib-uted studies elucidating small molecular requirementsof mammalian cells in tissue cultures utilizing massivecell inocula. 4 ' In studying the growth requirements ofindividual, isolated cells, we found that single cells ofthe S3 clonal strain of the HeLa cell form colonies with100% plating efficiency, when a chemically definedsmall-molecular medium is supplemented with twomacromolecular serum fractions each migrating as asingle moving boundary in an electric field. The twoproteins of mammalian serum which, under the condi-tions of these experiments, complete the growth require-ments of single cells are serum albumin and an o.~-globulin with a molecular weight of approximately45 000, which appears to be a glycoprotein in compo-sition. Both substances are necessary for growth ofsingle cells under the specific conditions of our pro-cedures. The functions of these proteins are still understudy, but that of the a~-globulin has been found to befulfilled completely by the protein fraction namedfetuin, an a~-globulin which constitutes 45% of calffetal serum protein. ~

    Albumin and fetuin have been purified by repeatedprecipitation to the point where preparations areapproximately 98% homogeneous electrophoreticallyand ultracentrifugally, although the ultimate purity ofsuch preparations is, of course, difficult to specify withcertainty, particularly since fetuin has been demon-strated to be heterogeneous immunologically. A typicalelectrophoretic pattern is presented in Figs. 3(a), and3(b) demonstrates the colonial development achievedfrom inoculation of single cells into a medium containingthe purified albumin and fetuin, amino acids, growthfactors, salts and glucose. The plating efficiency equalsthat in serum-containing medium. Experiments onmutants of S3 which exhibit different molecular nutri-tional requirements are now in progress.

    It seems likely that fetuin and albumin may not berequired as true metabolites, but rather as conditioningfactors which permit establishment of the necessaryphysicochemical relationships between the cell andsurrounding medium. Thus, the fetuin fraction exercisesa specific action on the cell membrane, since trypsinizedcells added to a medium, complete except for thepresence of fetuin, remain as rounded spheres, unat-tached to the glass surface. With the addition of fetuinto such a medium, the cells rapidly attach to the glassand stretch out in a highly characteristic configurationLFigs. 4(a) and 4(b)]. As little as 1 mg/cc of fetuin canbe detected by means of this action. Fetuin has beenknown to be a powerful antitryptic agent, and at leastpart of the function of fetuin seems to be antiproteo-lytic in nature, because this substance prevents thetryptic loosening and rounding of glass-attached cells.The participation in the attachment reaction of aprotein not included in the fetuin fraction has bt;enclaimed by some workers. '

  • T. T. PUCK

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    PxG. 3. (a) Klectrophoretic pattern of fetuin purified by repeated, fractional (NH4) 2SO4 precipitation from fetal calf serum. {b)Coloniesdeveloping on plate seeded with 100 S3 cells, in a medium containing known small molecular components, and purified fetuin and humanserum albumin.

    In further development of tools for quantitative studyof the growth and genetics of the mammalian cell

    in vitro, it became necessary to attempt to control thelability of karyotype which had been demonstrated toafI'ect cells cultivated by standard tissue-culture tech-

    niques. Recent studies have shown that virtually everymammalian cell which has been established as a stabletissue-culture strain has a chromosomal constitutionradically diGerent from that of the parental speciesfrom which it originated. ' "Euploid cells were generallyfound either to stop growth after a very short periodin culture, or else to proliferate, but with drastic changesin chromosome number and structure. In order toachieve conditions eliminating this proclivity for aneup-loid chromosomal constitution in cell cultures (whichrenders genetic studies of questionable significance),methodologies were required for simple and reliablemonitoring of chromosomal constitution of cells grownin vitro. Such methods were developed by Axelrad andMcCulloch, "by Rothfels and Siminovitch, "and by Tjioand the author in our laboratories. "%hen the karyo-types of such cultures could be checked with ease andaccuracy, it was possible to find growth conditionswhich permit karyotype integrity to be maintained.These conditions were secured by development of ascreening test for media and regulation of other condi-tions so as to remove conditions leading to mitoticinhibition, which might induce polyploidy. By careful&ontrol of the processes involved in cell dispersion, by

    regulation of the pH and temperature during cellincubation, and by use of pretested media whichincorporate all the needed nutritional requirements forcell growth and exclude toxic substances, it becamepossible to grow cells from normal tissues of man andother mammals, for periods now approaching a year,and for numbers of progeny which have exceeded 10"without change in chromosomal constitution. "Figure 5shows the chromosome complement of typical humanmale and female cells. These methodologies have per-mitted characterization of each of the human chromo-somes, and particularly of the X and Y sex-determiningchromosomes. '5

    In order to employ human genetic markers withbiochemical, immunologic, and pathologic character-istics, a method was devised by which cells from anyindividual could be simply and reliably introduced intostable growth in vitro. A small piece of skin, approxi-mately 10 to 50 mg in mass, is excised from the ventralpart of the forearm, dispersed by trypsinization, andgrown in the medium which contains fetal calf serum,which was shown to maintain stable karyotype. Thismethod has proved itself reliable in securing activelygrowing cultures from any individual. '4 It has madepossible confirmation of the fact that the humankaryotype does, indeed, consist of 46 chromosomes, asfirst reported by Tjio and Levan. " Studies are nowunder way on cells of individuals with phenyl ketonuriaand other genetic diseases, in an attempt to establish

  • STUDIES ON MAM MAL IAN CELLS IN VITRO

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    cytogenetic and biochemical differences between theseand normal cells in the irI, vitro cultures. These method-ologies are also being employed clinically to determinethe chromosomal constitution of persons with variousdegrees of clinical hermaphroditism and to comparethe results so obtained with those utilizing the sexchromatin method devised by Barr and his associates. "Thus, an anatomically female patient su6ering fromovarian dysgenesis (Turner's syndrome) was shown topossess only 45 chromosomes.

    In contrast with the normal human chromosomeconstitution shown in Fig. 5, Fig. 6 presents that ofthe aneuploid S3 HeLa cell, a clonal stock which wedeveloped from the culture which Gey and his associatesestablished from a human carcinoma of the cervix. "The chromosome number of this clonal strain is 78,which represents a hypotetraploid condition. Studiesare in progress comparing the diferent morphology andbehavior of cells which dier in chromosomal numberand constitution. Thus, mutant clones of animals likethe Chinese hamster have been isolated, which havestemline chromosome numbers of 22 and 23, respec-tively.

    As one example of the kinds of quantitative studiesmade possible by these techniques, the remainder ofthis discussion is devoted to analysis of their applicationto study of the action of high-energy radiation on mam-malian cells. Ionizing radiations are capable of disrupt-

    ing any chemical bond in any molecule of any cell whichhas absorbed such energy. On the basis of such qualita-tive considerations alone, one might expect an enormousvariety of cellular pathologic actions. Radiobiologicstudies on mammalian systems carried out by manylaboratories over many years have more than borne outthis expectation, demonstrating a bewildering variety ofpathological consequences attending various kinds ofexposure to radiation of mammals, including phenomenaas diverse as gastrointestinal disturbance, drop in thelevel of blood white cells, epilation, and carcinogenesis.One of the most critical problems in this field involvesdetermination of the degree to which chemical disorgani-zation attending exposure to a given dose of ionizing ra-diation aff'ects the genome or the phenome, respectively,of the irradiated cell. A quantitative answer to thisquestion would greatly simplify attempts to understandthe enormously complex series of events attending irra-diation of mammalian cells and of whole animals. Inaddition, the answer to this question is essential inorder to determine the degree to which exposure ofmammalian populations to various doses of ionizingradiations will result in random mutations transmissibleto succeeding generations.

    Physiologic damage to cells in the form of reversiblelag periods in cell multiplication, changes in cell perme-ability and inhibition of many specific enzymaticactivities, have been demonstrated in many organisms.

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  • STUDIES OX MAM MALIAN CELLS IX VITRO 439

    FIG. 6. Chromosome complement of atypical S39 HeLa cell, a subclone of theS3 strain, of human malignant origin.with a stemline number of 78.

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    In a variety of nonmammalian forms it had also beenshown that the cell nucleus is much more sensitive todamage by irradiation than the cytoplasm. Genetic andcytologic examination of radiation eBects, particularlyin the simpler organisms, has revealed that changes ofthe genetic structure arising from irradiation includesingle gene mutations, chromosome breaks, productionof areas deficient in chromatin, and occasionally elon-gated and amorphous chromosomes, these latter pre-sumably arising from cells which had been irradiatedduring mitosis. At the site of fragmentation, a chromo-some exhibits sticky surfaces which can re-attach sothat the chromosomes may reconstitute themselves intoa configuration more or less closely resembling thenormal. In all probability, however, a defect, eventhough invisible, will persist at the site of breakageunless this happened to lie in a genetically inactiveregion. All or part of any chromosome which hassu8ered such a "hit" may, through imperfect restitu-tion, fail to be incorporated in the mitotic apparatusand be lost in subsequent cell divisions. Such a conditioncan cause genetic imbalance that may destroy theability of a cell exhibiting it to reproduce indefinitely.

    In addition, if an unrestituted chromosome divides, thetwo sister chromatids may unite at their broken endsto form an anaphase bridge that can prevent completionof mitosis. If multiple chromosome hits occur in thesame cell, all of the foregoing damaging actions char-acteristic of single breaks are intensified. Moreover,abnormal restitution of the sticky, broken ends ofdiferent chromosomes may occur, producing a varietyof complex translocations, such as dicentric chromo-somes which can destroy the cell's reproductivecapacity. "

    Earlier studies of the relative sensitivities of themammalian genome and phenome, respectively, todamage by high-energy radiations at first seemed tofavor the thesis that for mammalian cells, in contrastto cells of other organisms, physiologic rather thangenetic e6'ects may be most important in the dose rangeup to and including the mean lethal dose for the wholeorganism, which is about 400 to 500 r. This suppositionappeared to be supported by several kinds of evidence:studies in which massive cell inocula were irradiated intissue culture with progressive doses of irradiationrevealed that permanent loss of cell proliferation did

  • 440 T. T. PUCK

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    Fro. 7. X-ray survival curve of the ability of single S3 HeLacells to form colonies as a function of the dose, D. The points canbe fitted either by the equation N/NO=1 [1e ~")' or byN/=e ~"(1+8/77), the latter relationship being preferable,since it is based on the model that reproductive death for thispolyploid cell requires two or more effective hits anywhere in thechromosome complement. A more refined and comprehensiveequation which expresses the contributions to genetic death ofhaploid, diploid, or polyploid cells of any species by single-hit andmultiple-hit radiation damages is presented elsewhere.

    not occur until many thousands of roentgens had beenadministered. Presumably because of the way in whichtissue-culture thinking in the past was dominated bythe concept that cell proliferation is a property of acellular community, experiments like this were inter-preted as an index of the dose needed to inactivate thecellular reproductive mechanism. Moreover, irradiationof single cells of bacteria, yeasts and protozoa, whichmight be considered as models for the mammalian ce)l,indicated that in all these forms, inactivation of repro-duction required thousands of roentgens. "Hence, sincedeath of a whole mammal can be eGected by an exposureof only 400 to 500 roentgens, it appeared possible thatmammalian-cell interaction with irradiation proceedsin a manner fundamentally diGerent from that of otherforms. While this thesis has been vigorously opposed,especially by geneticists, among whom H. J. Mullerhas been particularly active, it is still occasionallyalarmed in the current scientific literature that veryhigh doses in the neighborhood of many thousands ofroentgens are needed to achieve irreversible damage tomammalian cells in eitro20 and the ability of ionizingradiation to produce mutagenesis in mammals is stillbeing challenged. "

    Kith the development of the techniques which havebeen here described, it became evident that the use ofcell proliferation from massive cultures cannot be usedas an end point to quantitate irreversible effects ofradiation on cell reproductive capacity unless the resultsare calculated as a survival function based on thenumber of cells irradiated. Since even a single cellremaining intact can proliferate eventually to produceany degree of outgrowth whatever, the dose at which alarge but unspecified population fails to producerecognizable outgrowth is meaningless in terms of thedose needed on the average to inactivate the cellularreproductive apparatus. Moreover, since it is character-

    istic of irradiated cells to exhibit reversible mitotic lags,and to continue to multiply for one or even severalgenerations, but then to produce a microcolony of sterileprogeny, use of the mitotic index of the population asa measure of irreversible eGects on cellular reproductionis not reliable. However, the use of the single-cellplating procedure makes possible precise determinationof survival curves for mammalian cells by proceduresexactly like those which had been used for cells ofE. col~. In this way it is possible to measure accuratelythe dose required for inactivation of the reproductivemechanism of the individual cells constituting a popu-lation. The first such curve obtained is shown in Fig. 7,and approximates a two-hit relationship, with an initialshoulder, followed by a linear exponential drop, whichis maintained for doses up to at least 1800 r. Of majorimportance is the fact that the mean lethal dose, Do,which is obtained from the slope of the linear part ofthe curve, was only 96 r. The determination of thiscurve is completely objective and has been verified bythis time in a number of laboratories.

    Several features of the behavior of such irradiatedcells pointed to damage to the genome as the primaryprocess responsible for reproductive death. "The smallvalue of the mean lethal dose for the S3 HeLa cell madeit possible to rule out of consideration single-genemutation as the dominant radiobiological process. Thehypothesis was advanced that cell death is a conse-quence of chromosomal damage resulting from irradi-ation, and this proposal appeared to give good quanti-tative fit with the behavior of the system. This inter-pretation was supported by the following facts. Afterx-irradiation of single cells with approximately 6 meanlethal doses, the survivors were allowed to form colonieswhich were then picked and grown into new cell stocks.Examination showed at least 4 out of 5 such strains tobe mutated from the original form, exhibiting morpho-logical or nutritional diGerences, which persisted afterlong-term growth. Moreover, each of these four strainswas revealed to possess a chromosomal constitutiongrossly altered from that of the original, unirradiatedstrain (Fig. 8). The 2-hit nature of the curve for theS3 cell was explained on the basis that reproductivedeath in such cells requires, in the main, at least twoindependent hits, and therefore appears to be a resultof aberration produced by interaction between twoseparately damaged chromosomes. Other aneuploidcells of human origin, like S3, and with chromosomenumbers far in excess of the normal 46, gave x-ray sur-vival curves similar to that of S3."

    The fate of the cells whose ability to multiplyindefinitely has been destroyed by irradiation is ofinterest. While some may multiply for a few generations,all appear to retain the ability to metabolize eGectivelyand to carry out complex biosyntheses of macro-molecular structures like that of a specific virus, addedto the system after irradiation. Under proper environ-mental conditions, cells of either aneuploid or euploid

  • STUDIES ON MAMMALIAN CELLS IN VITRO 44i

    constitution may, after irradiation, produce giant forms.Thus, these cells which have been destroyed reproduc-tively by irradiation, have maintained intact a largeproportion of their other metabolic activities. At leastsmall distortions of these functions probably also exist,but may require subtle means for their demonstration.

    A pattern of events similar in some ways, but differentin others, was obtained on irradiation of human cellswith normal chromosome complements. All of thesecells originated in normal human tissues and possessedthe typical, elongated configuration commonly associ-ated with euploid human cells." Determination ofchromosome constitution on several of these revealedthem to have the normal diploid number. All of thembehaved identically, and exhibited a mean lethal doseof about 50 r, corresponding to an even greater radio-sensitivity than the HeLa cell. The hit number of thesesurvival curves is less than that of the aneuploid HeLacell, and lies somewhere between 1 and 2, a greateraccuracy not yet being available for these cells becauseof their great radiation sensitivity. Hence, the conclusionmay be drawn that euploid human cells are even moreradiosensitive than the malignant, aneuploid HeLa.

    As a further test of the hypothesis that cell repro-ductive death, as a result of x-irradiation, arises directlyfrom chromosomal damage, a series of experiments wascarried out in which euploid human cells were irradi-ated, after which the chromosomes were delineated andexamined cytologically for direct evidence of aberra-tions. A preliminary report by Bender, '4 counting

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    TABLE I. Chromosome aberrations at various radiation doses.Single-hit aberrations are chromosomal defects caused by a singleionizing event, and include a complete break in one chromatidonly; a break in both chromatids at the same point presumablyreflecting a break in the chromosome before it has doubled or atraverse of both chromatids by the same ionizing particle; anachromatic region in which the continuity of the chromosome isuninterrupted, but chromatin has disappeared from a particulararea; or the presence of one or more greatly elongated chromo-somes trailing "sticky" streamers. Multihit complexes comprisechromosomal aberrations involving interaction between two ormore independent hits to 1 or more chromosomes, such as trans-located dicentrics and ring chromosomes.

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    chromosome breaks induced in euploid human cellsgrownin vitro, suggested that approximately 300 r wererequired to produce an average of one visible chromo-some break per cell. This figure is almost six timeshigher than the mean lethal dose for these cells. In anattempt to obtain an estimate of the roentgen eSciencyof chromosome breaks for euploid human cells, validunder our conditions of study, experiments were carriedout in our laboratory in which the conditions of growthof the cells were brought as nearly as possible tooptimal values in order to minimize some of the uncer-tainties owing to mitotic lags. In addition, however,an independent method of arriving at the chromosome-breaking eKciency was employed, based on the factthat the appearance of aberrations due to abnormalchromosome restitutions, can occur only in appreciablenumbers at doses beyond the mean lethal dose. Suchabnormal restitutions will not disappear, as can singlechromosome breaks which can reseal without leavingany visible trace."

    Figure 9 presents typical sets of mitotic figuresobtained when cells with normal chromosome consti-tution taken from various tissues of normal humansubjects are irradiated in vitro at different dose levels.It is obvious that, with cells irradiated with 50 r, singlechromosome breaks are readily evident LFig. 9(b)j.When the dose is increased to 75 r, two effects appear:the number of single breaks is increased; in addition,however, new types of aberrations appear which indi-cate that, at this dose, multiple chromosome hits inindividual cells have become a frequent occurrence.Examples of such complex anomalies formed throughinteraction of several radiation-damaged chromosomalsites are presented in Figs. 9(c) and 9(d). A summaryof this data is presented ig. fable I. From these figures,

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    p~/4

    +4' 5

    FH:. 9. Typical kinds of chromosome abnormalities observed when euploid human cells are irradiated ie nitro with 230-kv x-rays.(a) Unirradiated cell. (b);Cell irradiated with 50 r. Breaks and deletions appear as indicated by arrows. (c) Chromosomes of cell irradi-ated with 75r, showing a translocation in the center, and a dicentric. (d) Portion of a mitotic figure of a cell after f50-r irradiation,demonstrating formation of ring chromosomes.

  • 444 T. T. PUCK

    one can safely conclude that the dose needed to intro-duce an average of one visible chromosome break percell is no more than 40 to 60 r, a value which agreeswithin the limits of experimental uncertainty with thatobtained as the mean lethal dose for colony formationof these cells.

    The conclusion may be drawn that the genome of theeuploid human cell is extraordinarily susceptible tochromosome damage by ionizing radiation, and that theaverage dose needed to introduce one chromosomebreak per cell is similar to the mean lethal dose to thecell's reproductive function, i.e., approximately 50 r.Since this value is far less than that required for theinactivation of various enzymatic activities in mam-malian cells,"it appears to establish the fact that thecellular genome of man is far more sensitive to radiationdamage which is, at least in part, presumably irrevers-ible, than are other of its structures. While studies arecurrently in progress to determine whether cells in vkowill behave in the same way, experiments with radio-protective agents added to the medium suggest thatonly relatively small changes in the cellular Do value canbe accomplished by changes in the cellular environment.

    These considerations afford an understanding of theaction of ionizing radiation on mammalian cells in termsof a primary effectthe initiation of a chromosomebreak, which requires on the average an exposure of anormal human cell to 50 r or less, and a secondaryeffectthe production of complex chromosomal aber-rations through abnormal restitution of the fragmentsresulting from multiple breaks occurring within thesame cell. The erst of these involves a variety ofalternative possibilities: The cell may continue repro-duction with no recognizable cytogenetic change, thoughwith perhaps a gene mutation at the site of the originalchromosome break; it may suffer loss of all or part of achromosome which could impair its ability to reproduce;or it may undergo formation of a chromosome bridgeat anaphase which will certainly end its reproductiveability. When multiple chromosome fragmentationsleading to complex translocations occur, the cell is muchmore likely to be incapacitated with respect to repro-duction. Different cells should display i-hit, multiple-hit, or intermediate types of survival curves, dependingon the degree to which these different processes operatein causing destruction of the ability to multiply.

    This analysis appears to offer an explanation for thedifference in radiobiologic response exhibited by variousmammalian cells. For example, normal diploid cells ofdifferent species, differing in their total mass of chromo-some material, should be expected to exhibit differencesin radiation sensitivity roughly though not exactlyparalleling their chromosomal volumes, since the cellwith greater chromosomal mass will offer greater oppor-tunity for a given radiation dose to produce ionizationdirectly or indirectly effective within this region. Inaccordance with this expectation, we have found that

    TAar.x II. Comparison of radiation and the DNA content fordiploid cells of three different living forms. The data indicatethat, despite individual variations in Do values and DNA contentsof over a hundred-fold, the product of these two variables remainsreasonably constant, varying only by a factor of about two.

    Dip-loidcell

    Dp value'

    Product:DNA Dp XDNA

    content content(in picograms (roentgens

    per cell) Xpicograms)

    YeastChickMan

    about 8000 rbabout 300-400 r'50-60 r'

    0.05b2.5~8.3d

    400900500

    The Dp value is taken as the dose needed to reduce the reproducingfraction of the cell population to 37%, in the region where the survivalcurve is linearly exponential.

    b See reference 27.p See reference 25.~ See reference 28.e See reference 22,

    cells of the chick, whose DNA content is less thanone-quarter that of human cells, possess a mean lethaldose for x-rays approximately 4 to 8 times greater thanthat of euploid cells of man. In Table II is presented acomparison of the Do values and DNA content ofdiploid cells from a variety of different species. Despiterelatively large uncertainties in the individual determi-nations of the mean lethal dose and of the DNAcontent, and the large range of values encompassed, itis evident that these very different diploid cells exhibita remarkably constant value for the product of these 2variables. Other aspects of these relationships areconsidered elsewhere.

    These considerations afford insight into the effectsof various chromosome conditions like polyploidy onthe radiosensitivity of mammalian cells from the samespecies. With increasing numbers of chromosome setswithin a cell, its sensitivity to killing by a 1-hit processshould decrease. Thus, a haploid cell will lose its abilityfor indefinite multiplication as a result of damage toany single gene essential to replication. Diploid cellswhich have a double gene set presumably will requireloss or aberration of larger portions of a chromosomebefore the genetic imbalance necessary for inhibitionof multiplication results. Since such chromosome lossesreadily occur as a result of 1-hit processes followingexposure to ionizing radiations, such cells will oftenexhibit curves with a hit number close to unity, butmay in some cases be multiple hit, if the gene distribu-tion among the chromosomes is such that death rarelyfollows a single-hit process. The Do value of such curveswill be influenced by the volume of each chromosome,the magnitude of the breaks which are produced, andthe degree to which 1-hit lethal and 2-hit lethal processesoccur when cells are exposed to various types of irradi-ation under various conditions. Cells with higher degreesof ploidy will be much less susceptible to reproductivekilling by loss of part or all of any chromosome becauseof the smaller imbalance produced when larger multiplesof the chromosome set are present. In:such cells, themajor lethal process will involve interaction between

  • STU D I ES ON MA M MAL I AN CELLS IN VITRO 445

    two or more chromosomes to produce malformationslike anaphase bridges. Hence, such cells will usuallydisplay a hit number in the neighborhood of 2. Whilethe larger volume of the chromosome set in such cellsmight at first be considered to make them more vulner-able than diploid cells to the accumulation of hits thatcan lead to cell death, this factor may be more thancounterbalanced by the relatively low probability withwhich two simultaneously broken chromosomes willrestitute abnormally, so as to form a nondividingconfiguration (like a dicentric) as opposed to a unionstill permitting normal mitosis. Thus, the HeLa S3cell, an aneuploid human malignant cell with 78chromosomes, exhibits a survival curve which is approx-irnately 2-hit, as opposed to the more nearly 1-hitcurve of the euploid cell, and exhibits a Do value of96 r instead of 50 r characteristic of the normal diploidcell. These parameters the degree of cell ploidy; thedistribution among the different chromosomes of regionswhose loss may lead to cell death through imbalance;and the separate probability of formation of chromo-somal aberrations which mechanically prevent un-limited division by 1-hit and 2-hit processes distributedamong the various chromosomes would appear toafford explanation of the difference in radiosensitivitiesof different cells. Quantitative evaluation of these pa-rameters in selected normal and malignant cell systemsis now under study.

    These considerations also can offer explanation forour recent findings that the S3 cell which is enormouslymore sensitive than bacterial cells to killing by x-rays,is much more resistant to ultraviolet irradiation. Ultra-violet, while a highly effective lethal and mutagenicagent for bacteria, is known to be much less efficientthan x-rays in producing chromosome breaks. Whilehaploid bacterial cells can be killed effectively by pointmutations and so are readily inactivated by ultravioletlight, the need for chromosome breakage to occur beforethe multiploid animal cell is inactivated would renderit resistant to the action of ultraviolet irradiation.

    Application of these aspects of the action of ionizingradiation at the cellular level, to analysis of the under-lying mechanism involved in the pathologies arisingfrom acute, whole body irradiation of animals leaveslittle doubt that many of the most important featuresof the mammalian radiation syndrome are due to thecumulative actions on the individual body cells of thesame kind as those described in the preceding para-graphs:

    (a) Knowledge of the Do value for individual cellsfor the first time provides clear explanation why themean lethal dose for total body irradiation of mammalsshould lie in the range of 400 to 600 r. Exposure of thewhole body to a dose of this magnitude would inactivatethe reproductive mechanism of more than 99% of thecells with radiosensitivities like those of the cells studiedin ~pro. The body might recover from smaller doses

    which would still leave sufficient intact cells whichcould reproduce at a rate rapid enough to compensatefor the losses. The mean lethal dose to the whole animalrepresents a point of loss of reproducing cells so exten-sive as to provide widespread damage to the integritiesof physiological function which depends on cell repro-duction and which constitutes a stress which cannotordinarily be withstood.

    (b) This formulation is in accord with the failure tofind any specific biochemical lesion resulting fromanimal irradiation in the mean lethal range. Significantdepression of individual enzyme functions have beenfound to require much higher doses than that neededto kill the whole animal. While no specific enzymefunction could be implicated, many studies showedthat DNA synthesis was greatly reduced as a result ofirradiation in the dose range here considered. " Thetheory here considered would predict such behavior.DNA synthesis can proceed normally only when cellreproduction progresses. Destruction of this functionthrough production of chromosome aberrations musteventually depress DNA synthesis, even though noenzyme system of the cells or body fluids has beensignificantly altered by the irradiation.

    (c) Similarly, the failure to find any accumulation oftoxic material in the body fluids after exposure toradiation in the mean lethal range is to be expected.While at higher doses significant amounts of toxicproducts might be produced as a result of chemicalstructural alterations resulting from ionizations pro-duced in the tissue, doses of several hundred roentgensonly will alter significantly structures with a size of theorder of magnitude of the chromosomes, and willproduce biological changes only if the uninterruptedintegrity of such structures is vital to normal bodyoperations as is indeed the case with the geneticapparatus of the individual cells.

    (d) It follows that one would expect those tissues tobe most radiosensitive which must maintain the highestrate of cell mitosis, since these will suffer the mostimmediate loss of their specific functions, on which thebody depends. This correlation between radiosensitivityand mitotic rate has been recognized as one of theearliest generalizations to emerge from radiobiologicexperience. However, while the rapidly dividing tissuesare indeed most radiosensitive from the point of viewof their immediate contribution to embarrassment ofthe whole organism through failure to maintain theirspecific functions, it is necessary to regard virtually allof the body cells as equally sensitive to chromosomaldamage. Each normal cell nucleus has an equal proba-bility of accumulating similar chromosomal injuries.Such aberrations will remain largely latent in each celluntil it comes to reproduce. Hence, the cells of themore rapidly dividing tissues are simply the first tomake evident the injuries which must be regarded asequally numerous in the more slowly multiplying

  • T. T. PUCK

    regions of the body. This fact is of vital importance inunderstanding genetic transmission of radiation dam-

    age, and the ability for tumors to develop many yearsafter a radiation experience. ~

    (e) Similarly, our picture accounts in straightforwardfashion for the characteristic lag period between radi-ation exposure and the development of symptoms,which has long been recognized as typifying mammalianinjury by ionizing radiation. The cell which has suffereddamage which is primarily only chromosomal, willcontinue to function effectively for a period, since itsfull complement of enzymes and other structures pre-sumably remains largely unaltered. Not until the cellreaches the point where it should normally divide willit begin to exhibit grossly deviant behavior from thenormal. Moreover, we have shown that such cells mayeven multiply for several generations, before they andtheir progeny cease reproduction. "

    (f) The sequence of events which ultimately leads todeath of any acutely irradiated animal may thus beexplained as follows. Chromosomal aberrations intro-duced among all of the cells exposed will first revealthemselves in those cells of the most actively mitotictissues. Division will be prevented in most of such cellsirradiated with several hundred roentgens, with theresult that the functions supplied by these tissues willfail and the body will be threatened. As other more-slowly dividing tissues reach the point where they tooshould produce cell proliferation, they will add theresults of their failure to those which have alreadyintroduced distortion of normal body physiology. Incontrast to the actively dividing structures like thebone marrow, and the epithelial linings of the gastro-intestinal tract, tissues like nerve and muscle in whichcell division rarely occurs, can continue to exhibitnormal function even after exposure to many thousandsof roentgens. It is of interest that cell reproductivedeath has also been identified as the major factor in themammalian radiation syndrome by Quastler and hisco-workers, using experimental approaches and tech-niques completely different from those employed here. "

    In this connection, an alternative hypothesis hasbeen proposed that the more-rapidly dividing tissuesare more radiosensitive only because cells in mitosisare in a more sensitive condition and hence are damagedmore readily by irradiation. This proposal does notfit the facts, since even in the most rapidly dividingtissues, no more than 3% of the cells are in mitosis atany one time. Hence, if this were the true explanation,it could account at most for a negligible reduction incell viability of such tissues. However, in early embry-onic development, where high mitotic frequencies oftenare achieved and where phased cell reproduction mayoccur to bring many cells into mitosis simultaneously,this factor might easily play an important role.

    (g) These considerations also explain quite naturallyhow it is possible to save animals irradiated with doses

    TABLE III. Data demonstrating that possibly some parallelismmay exist between the mean lethal dose for whole warm-bloodedorganisms (LDf;0) and the Do values of their single, euploid cellsas determined in vitro.

    X-ray LD&o forwhole animal

    X-ray D0 of euploidcell f',I vitro

    ManChinese hamsterFowl

    400- 500 r8251190 r'

    1000 rb

    5060 r160 r

    300-400 r

    See reference 33.b See reference 34.

    in the mean lethal range, by injection of viable bone-marrow cells, though not by cellular fragments orextracts. Such cells recolonize the irradiated tissuewhich is being depleted through impaired cell-repro-ductive function. While other tissues have presumablysuffered equal diminution in the percent of cells capableof reproduction, these require replenishment less rapidlyand hence provide more time for the surviving cells torecolonize the tissue, provided the bone-marrow func-tions can be maintained. It may be expected thatanimals might be saved from lethal effects of evenhigher doses, by additional inoculation of viable cellsfrom other tissues which, as the dose increases progres-sively, would become critical in their failure to maintainfunctional integrity of the body.

    (h) Similarly, the interesting experiments first carriedon by Patt and his co-workers, "in which animals weresubjected to low temperatures immediately after irradi-ation, become explicable. Such animals failed to developany of the symptoms of radiation injury until aftertheir temperature was restored, after which the entiresequence of pathogenesis was initiated as though theirradiation had just occurred at the time their bodytemperatures had been raised back to normal. At thelow temperature, mitosis is inhibited so that the bodydoes not produce the conditions which can bring intoexpression the latent damage to the cellular geneticapparatus. On rewarming, normal cell reproduction isagain initiated, so that each time a genetically damagedcell comes to mitosis its injury becomes functional andcontributes to embarrassment of a normal function.

    (i) One might expect, then, to find some rough corre-lation between the Do obtained for euploid cells ofdifferent animals as measured by the survival curveshere described, and the mean lethal dose for the wholeanimal. While the latter figure must, of necessity, beinQuenced by many complex interactions of an unpre-dictable kind, it is of distinct interest to find that sucha correlation is at least suggested by the small amountof currently available data, as shown in Table III.

    Studies of the action of radioprotective agents onx-ray survival curves of S3 cells are completely con-sistent with the interpretations here developed. Thecompound, 2-mercaptoethyl guanidine, which has beendemonstrated to raise the MLD for mice from 900 to amaximum of 1450 r,""also exercises significant radio-

  • STUDIES ON MAMMALIAN CELLS IN VITRO 447

    protection on S3 HeLa cells plated in euro. The shapeof the survival curve remains approximately 2-hit as itwas in the absence of the compound, but Do, the meanlethal dose for cell reproduction, is raised from 96 r toa maximum of 160 r, a value which agrees far betterthan perhaps could be expected with the degree ofprotection achieved in the whole animal. All of the dataon the kinetics of the radioprotective action of thiscompound on single plated cells are consistent with theinterpretation that the presence of this material lowersthe effective dose of x-rays which reaches the siteswithin the cell whose inactivation results in loss of theability to reproduce indefinitely.

    (j) Finally, these data support the cellular-geneticinterpretation of the great effectiveness of relativelylow doses of radiation in suppressing antibody formationin the mammalian body. The dose range needed toinhibit antibody production significantly lies in theregion of 50 to 150 r," a value which, through its closecorrespondence with the mean lethal dose for euploidcell reproduction, suggests this action to reflect theeffect of radiation in preventing multiplication ofantibody-producing cells. By contrast, specific macro-molecular biosynthetic mechanisms appear essentiallyundamaged even after irradiation of mammalian cellswith thousands of roentgens. '5 Thus, the great radio-sensitivity of antibody production suggests that themechanism of new antibody production involves theneed for cell multiplication, rather than simply antibodysynthesis by pre-existing cells.

    The fact that some mammalian cells exhibit survivalcurves for the reproductive function which are multiple-hit in character must not be taken as an indication that athreshold exists for the induction of mutations by high-energy radiations. On the contrary, all of the evidencehere presented leads to the conclusion that, while a celldisplaying a 2-hit survival curve does display a thresholdfor killing by high-energy radiation, the production ofsingle chromosome breaks, and the attendant alterationof genetic material at the site of such a break, goes oneven when doses insufhcient to kill are applied. Fromthe point of view of the entire organism and its progeny,it may be far better for any cell to be permanentlyinactivated by multiple chromosomal aberrations thanto suffer only a single chromosomal change, which thenmay be handed on to the offspring, usually as a recessivegenetic defect.

    The data here presented make possible an estimationas to whether background radiation may contribute tothe processes of aging in man. Since 50 r is the averagedose needed to produce 1 visible chromosome breakper cell by the techniques here employed, one may wellexpect that damage on a smaller scale which cannot beseen by ordinary microscopy results from even smallerdoses. Thus, one can calculate that, with a backgroundexposure of O.i r per year, in 70 years the accumulateddose is sufhcient to have produced gross chromosome

    damage in one-seventh of all of the body cells, andprobably more subtle effects in a much larger number.This would appear not to be a negligible process, andthus makes it probable that the gradual attrition ofbody functions which constitute aging, may be, to avery significant degree, the re8ection of accumulation ofcellular genetic injuries in cells and their descendantsoriginating from background irradiation. By extensionof the methodologies here discussed, it is proposed toattempt analysis of physiologic and genetic differencesin the behavior of cells taken from aging animals, andto examine as well the effects of the molecular constit-uents of body fIuids from such subjects on physiologicand genetic behavior of standard cell strains in vitro.

    Discussion of the use of quantitative methods inmeasuring mammalian cell growth and genetics wouldbe incomplete without at least mention of the out-standing development by Dulbecco and his associates'8in achieving a plaque technique for precise enumerationof single particles of mammalian viruses exactly as hasbeen current in bacteriophage studies. Thus, it becomespossible also to quantitate virus and cell interaction inanimal systems, so that one may expect equally pro-found insights to arise from such studies as has beenthe case in the interaction of bacteriophages with theirbacterial hosts.

    The thesis which has been the purpose of thisdiscussion is that newer developments promise toprovide well-defined systems for quantitative explora-tion of physical and physicochemical mechanismsinvolved in growth, genetics, and differentiation inmammalian-cell systems. There is every indication thatin the space of a few years it will be possible to come togrips with specific molecular aspects of these basicmechanisms in as intimate a fashion as is now currentin the biophysical approach to microbial systems.

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