The Cancer Cell in Vitro: a Review

The Cancer Cell in Vitro: A Review

J.PAUL

(Biochemistry Department, Glasgow Univervity, Glasgow, Scotland)

Cancer is clearly the result of a defect of the GENERAL BEHAVIOR OF CULTURED homeostatic mechanisms which maintain the bal NORMAL AND CANCER TISSUE ance among cells in an organism. Since the nature Organized and unorganized growth.â€”Certain of these mechanisms is not understood it is not sur commonplace phenomena of cultured tissues are prising that nothing is known of the lesion which probably relevant to the cancer problem. causes the disease. Recent research indicates that, In the first place, as is well known, if fragments whereas the reaction of the host may contribute to of tissue are explanted they immediately tend to the establishment and growth of a cancer, there is become disorganized as a result of cell migration no doubt that the primary lesion occurs in the cell. which follows a definite pattern. Cells of the reticu The tissue culture method has therefore been em loendothelial system, being most highly motile, ployed to study the cell lesion in isolation from escape first, but they rarely exhibit mitosis. A the host. wave of migration of fibroblasts follows, and these The implications of tissue culture in cancer cells begin to multiply as soon as they leave the research were appreciated very early, and in 1906 explant. If epithelial cells are present they migrate Beebe and Ewing (7) attempted to grow an â€œin later as a sheet, and mitosis may also commence in fective canine lymphosarcomaâ€• in the blood of in them. Finally, many of the highly specialized cells fected and uninfected dogs. Some of Alexis Car hardly migrate at all and may be recognized within rel's earliest experiments also included attempts to the thinned-out explant. When pieces of tissue culture cancer tissues and compare their behavior have become disorganized in this manner the with that of normal tissues. For technical reasons emigrating cells show little tendency to react with little of this early work provided useful informa one another. Cells from adjacent explants will tion, and perhaps the earliest significant observa migrate toward one another and will intermingle tion was Carrel's (11) finding in 1926 that the Rous intimately whether they are from the same or sarcoma virus would survive in embryo chick different organs. tissues in vitro. In the following years each succes In contrast, if tissue architecture is maintained sive theory made its impact on cancer studies in by careful manipulation and migration is dis tissue culture. Thus, Earle (@,3,24) and his col couraged, as is done in organ culture, then there leagues initiated investigations into the effects of is no tendency to become disorganized, and chemical carcinogens on cultured animal tissue, continuation of normal function can be convinc while Warburg's (92) hypothesis that cancer was ingly demonstrated (26, 81, 96). due to a lesion of the respiratory enzymes in the These simple observations themselves speak for cell was tested by Goldblatt and Cameron (34), some kind of homeostatic mechanism at the tissue and recently the virus theory has commanded level, and may be significant, as will be discussed most interest. later, that cancer cells, unlike normal cells, may The literature describing the use of tissue cul exhibit infiltration in organized cultures. ture in cancer research has already been admirably Primary explants.â€”It is now clear that cells reviewed in detail up to the year 1958 (48, 49, 63, growing out from primary explants do not undergo 64). The object of this article is to consider the any radical dedifferentiation. Cells in the migrat present situation critically and to suggest some ing zone tend to preserve something of their char conclusions which may be drawn. Only those re acteristic morphology, and in some instances this ports which are relevant to the discussion are men is of use in classifying and diagnosing tumors. For tioned, and recent review papers are referred to instance, Murray and Stout (66) have shown that where possible. an early diagnosis of sympathicoblastoma can be Received for publication September 25, 1961. made by tissue culture, since the neuroblasts send 431

out neurites which can often be seen in @4hours events is rather exceptional. More usually, after an and are almost always recognizable in 48 hours initial period of rapid growth the rate of multipli (Fig. 1). The same investigators employed tissue cation decreases, and the cells enter a more or less culture to help define the cellular origin of certain stationary state, when their survival may be in tumors such as the hemangiopericytoma (glomus doubt. Most cultures then die out, but at some tumor) (Fig. @),liposarcoma, and rhabdomyo time during this period there may be a sudden out sarcoma. burst of growth at several places in the culture. Apart from these specific morphological fea This appearance of a rapidly growing strain of tures there seem to be no general properties which cells from an otherwise slowly growing culture is distinguish normal cells from tumor cells in pri referred to as â€œtransformation.â€•It is of particular mary explants, with one possible exception. This interest in a discussion of the cancer problem, since concerns the phenomenon of surface inhibition it is associated with changes which resemble (2), which consists of a momentary cessation of malignant changes. surface movement when two normal fibroblasts Several mechanisms have been considered (apart from the simulation of transformation by Passaqe Number of chromosomes accidental contamination of a primary culture NÂ° 2x 45 with an established strain). It probably involves the selection of cells which are particularly suited to the environment. These favored cells may al ready be present at the time of isolation, or they may arise in some cases as a result of mutations or the redistribution of genetic material. Again, it has been suggested that the period of delay may

@â€˜ simply be necessary for the induction of some enzymes. Whatever the cause it is the manner in 2' which a cell strain usually arises, and the strain :@ which results will usually grow indefinitely in cul ture if properly handled. Cell strains.â€”Established cell strains resemble one another in many respects, as one might expect since the conditions of cell culture are highly selective. Nevertheless, three general morpho logical types are readily distinguishedâ€”namely, 40 50 60 70 60 90 fibroblastic, epithelial, and lymphocytic; and the CHART 1.â€”The development of aneuploidy and malignancy practised observer can often recognize further dif (tested by reinoculation) during the â€œtransformationâ€• of a ferences among individual lines. The principal culture of normal embryonic mouse skin cells. From Levan (52) after Levan and Biesele (58). (Reproduced by courtesy points of resemblance are similarities in growth of Dr. Levan.) rates and nutritional requirements (19) , the capacity to grow in relatively anaerobic conditions come into contact. Abercrombie and Ambrose (1) with a high rate of aerobic glycolysis, and the found that it was not displayed by Sarcoma 37 or tendency to develop unusual chromosomal com Sarcoma 180 cells, either toward similar cells or plements. toward normal cells cultured simultaneously. In Of these perhaps the most interesting is the view of the variation in behavior among similar tendency to develop chromosomal abnormalities. cells, it might be premature to draw any general This has been studied in detail by Levan and conclusions from these observations, but they are Biesele (53) and Hsu and Klatt (40), and they of such importance that they clearly merit repeti have pointed out that the first tendency is for tion and extension to other cell types. tetraploid cells to accumulate in the population It should be emphasized that the belief that (Chart 1). Later cells with a chromosome number cancer tissues grow more readily in culture than between diploid and tetraploid appear, and do normal tissues is a fallacy. In fact, Gey (32), eventually cells with a typical number of chromo from his extensive experience, indicates that the somes, very often in the triploid range, predomi opposite is often the case. nate. Ultimately, both normal and tetraploid cells

@ â€œTransforma/ion.â€•â€”The emergence of a cell may disappear entirely. The general result of this strain from a primary culture may occur without process is that almost all cell strains have an un any change in growth pattern, but this course of usual complement of chromosomes. The few excep

tions are nearly all skin fibroblasts, which tend to TRANSPLANTABILITY OF CELL retain the normal diploid number for reasons STRAINS OF NORMAL AND which are not clear. Chromosomal variations al TUMOR ORIGIN most certainly arise as a result of nondisjunction Coriell, McAllister, and Wagner (13) reported during mitosis, and they probably persist because that of a number of cell strains only HeLa cells they provide the cells with a selective advantage produced invasive metastasizing tumors in corti in the abnormal environment of a tissue culture sone-treated rats. On the other hand, Moore (59) medium. found no clear-cut difference between cell lines of There have been persistent suggestions that normal and tumor origin in similar experiments. these chromosomal changes may reflect the onset The discrepancy may be explained by the findings of malignancy Levan and Hauschka (54) pointed of Foley and Handler (28, 29) (Table 1), who in out that in a series of ascites tumors the chromo oculated known numbers of cells into the cheek some complement was almost never the normal pouches of normal and cortisone-treated golden diploid number. This observation led them to pro hamsters. They found that in cortisone-treated pound the stemline theory, which proposes that as the cells in a tumor escape from normal controls TABLE 1 they develop the capacity to adapt genetically and TUMOR FORMATION BY CULTURED CELL acquire the chromosome complement most suited LINES INOCULATED INTO THE to survival in a given environment. This theory [email protected] CHEEK POUCH was reinforced by observations of Levan and Foley and Handler (29) Biesele (58) in tissue cultures. They found that the establishment of an unusual chromosomal mode Type of Hne@ in which tumors was frequently associated with the onset of animal cells in were obtained with this malignancy, as tested by reinoculation (Chart 1). inoculumCortisone-treatedinoculatedNo. ocnlatedCell minimum Other workers have, however, failed to observe a HeLa, S-180 (All ma correlation between the onset of malignancy and hamsters10 lignant origin). chromosÃ mal variation. For instance, Hsu and 102 10@ Embryonic intestine. Klatt (40) showed that changes in chromosomal liver.Normal 10@KB, Normal complement of cultures of the Novikoff hepatoma were actually associated with loss of malignancy hamsters102 HeLa, J-111, Wilms 6, 5-180 (All of nialig and transplantability. The evidence now suggests nant origin). that chromosomal variation occurs in almost all 10@ Normal human amnion, established cell lines but is not necessarily associ embryonic intestine. 106KB, Normal human liver, ated with the onset of malignancy. lungs, thymus, endo The possibility remains that specific chromo metrium, normal mouse lymph-node, strain L somal abnormalities may be associated with the 929, normal monkey kid onset of malignancy. For instance, Nowell and ney. Hungerford (68) have observed that in human chronic myeloid leukemia blood cells character istically carry a minute chromosome which Baikie animals they could regularly produce tumors with et al. (6) consider to be a deletion of the long arm all cell lines, including those of normal origin, of chromosome 21 or 22. Similarly, Yerganian, when inocula larger than 10,000 cells were used. Leonard, and Gagnon (100) have found that the In the normal hamster tumors could regularly be onset of malignant transformation in vitro (as produced with all cell lines with inocula of more tested by reinoculation) in the cells of the Chinese than one million. However, when the inocula were hamster is associated with alterations in the reduced below these levels only cell lines arising morphology of the X1 chromosome. Clearly, from malignant tissue would produce tumors. For chromosomal rearrangements in cell strains may instance, the KB, HeLa, and S-180 strains pro or may not result in changes in a specific chromo duced tumors with inocula of less than a thousand some which may be important in tissue homeo cells in cortisone-treated hamsters and with stasis. Hence, unless each chromosome is individu inocula as low as 10,000 cells in normal hamsters. ally studied the significance of the observations Furthermore, when the tumors were allowed to may be missed. Incidentally, it is possible that grow for 9 days, those derived from â€œmalignantâ€• these observations may provide visual evidence for cell lines were usually healthy and uninfiltrated the deletion hypothesis, which will be referred to by host cells, whereas those originating from later. â€œnormalâ€•celllines were localized and infiltrated

by inflammatory cells. These observations mdi the cell. Similar observations were reported by cate a fundamental quantitative difference be other workers employing cultured normal and tween normal and malignant cells in their rela tumor tissues (17, 18, 43, 95). When later investi tionship with the host and are strictly in line with gators re-examined this question, however, several observations made with transplantable tumors by reports appeared claiming that many tumors Andervont and Shimkin (5). produced little or no lactate and sometimes non Southam, Moore, and Rhoads (82) undertook tumor tissues actually had higher rates of aerobic to inoculate human tumor cell lines into human glycolysis than did tumor tissues cultivated in volunteers, consisting of a group of healthy mdi vitro (30, 44, ,55, 90). These and other discrepancies viduals on the one hand and a group of cancer stimulated some investigators to question War patients in the last stages of the disease on the burg's theory and culminated in a public debate other. The healthy volunteers displayed a much by Weinhouse, Warburg, Burke, and Schade in the columns of Science (93). A clue to the nature TABLE 2 of the discrepancy was obtained by Paul and EFFECT OF PH ON THE CARB0. Pearson (72), who found that the metabolism of HYDRATE METABOLISM newly explanted normal tissues could fluctuate OF HIM CEu@s widely within the course of a few days. A similar observation was made by M. Harris (37) and later acidPHformed/glucoseLactic by others (4, 16, 31, 91). Recent studies (14, 69, 71) have demonstrated that several factors, es used6.60.06.80.027.00.067.20.27.40.827.60.57.80.758.00.85 pecially the leakage of intermediary metabolites from cells, the pH and glucose concentration in the

TABLE 3 EFFECT OF LOWERED OXYGEN TEN. SION DURING CULTURE ON REsPIRA TION SUBSEQUENTLY MEASURED IN Am (@l02 used/hr/cell X 10') higher resistance to the development of tumors at the site of inoculation. CENT OXYGEN IN GASPHASE0 These experiments indicate both that cultured Cai.z TYPEPER cells of tumor origin are more easily transplanted to foreign hosts than cells of normal origin and 20StrainL that the successful establishment of tumor cells on 7.32 inoculation depends to some extent on the state HeLa 7.60 11.00 3.91 5.62 of the host. HLM1.88

METABOLIC BEHAVIOR OF NORMAL medium, and the oxygen tension and carbon di AND MALIGNANT CELLS oxide tension to which cells are exposed during The metabolic behavior of cancer cells is of cultivation can all result in radical changes in the special interest, because rational attempts at metabolic patterns observed (Table 2). The most chemotherapy may be based on discovering funda interesting observation in relation to Warburg's mental differences between them and their normal hypothesis was that the metabolic pattern die counterparts. The observations which have corn tated by cultivation in a given environment per manded greatest interest in this field were made sisted for some hours after the stimulus was re by Warburg, Posener, and Negelein (92), who re moved (Table 3). Consequently, cells grown in a ported that cancer cells had a higher rate of rather low oxygen tension show a higher rate of aerobic glycolysis than did normal cells (i.e., when glycolysis and a lower rate of respiration in the incubated in an atmosphere of air they produced a usual experimental conditions. Similar observa larger amount of lactic acid and used more glu tions have been made very recently (3) by another cose) . These workers also found that frequently group. Since a low oxygen tension is character cancer cells had a lower rate of respiration. On the istically found within tumors Paul (71) suggests basis of these observations, Warburg propounded that these observations provide an adequate ex his well known theory that cancer was due to an planation for the rate of aerobic glycolysis in irreversible lesion of the respiratory mechanism of tumor cells.

In a few other respects significant metabolic dif Two interesting examples have been reported by ferences have been claimed between normal and Sanford et a!. from Earle's laboratory. The first malignant cells. In particular, Hirschberg and his concerns the strain L cell (which was originally colleagues (39) showed that azaguanine deami isolated from the C3H mouse and treated with nase occurred in very small amounts in human methylcholanthrene). At first this cell line pro glioblastoma but was present in rather large duced tumors in a high percentage of C3H mice, amounts in normal human brain. Unfortunately, but after some years of cultivation the incidence when an attempt was made to exploit this obser of tumor production fell. Perhaps the most inter vation in the treatment of patients with glioblas esting case of all, reported from the same labora toma no significant chemotherapeutic effect was tory, concerns a line of subcutaneous fibroblasts obtained. Another instance of a difference of this derived from C3H mice. The line was at one stage kind was claimed by Jacquez and his colleagues cloned (i.e., a single cell was isolated, and all sub (42), who found that a transaminase was absent in sequent cultures were derived from this). Two some mouse tumors, although it was present in divergent sublines eventually emerged, one of comparable normal tissues. This observation has which produced a very high incidence of tumors on not been applied in chemotherapy. Jacobson (41) re-inoculation into mice, whereas the other pro has demonstrated a difference between normal and duced very few. leukemic blood cells in their metabolism of folic De Bruyn also observed a loss of malignancy acid which may be exploitable. A somewhat differ during prolonged cultivation. Her original MB ent kind of differential effect has been reported by strains produced tumors in a large proportion of Leslie et a!. (51) in the effect of insulin on carbo inoculations, but some sublines subsequently de hydrate metabolism. rived from them were incapable of producing any Some interest attaches to the recent demonstra tumors at all. tion (94) that certain enzymes involved in the It is clear from this work that cells in continu synthesis of DNA increase greatly in amount be ous cultivation can spontaneously develop or lose fore a resting culture begins to grow and remain at the capacity to produce cancers. high levels until growth ceases. The addition to Tumor-producing viruses.â€”Carrel (11) observed the medium of the substrate (thymidine) for some that the Rous virus could be propagated in chick of the enzymes stimulates a further increase. embryo tissue culture, and Halberstaedter and These observations provide strong evidence for Doljanski (36) and Manaker and Groupe (56) the existence of feedback mechanisms of the kind made similar observations. The problem was taken suggested by Potter and Auerbach (74) and simi further by Rubin and Temin (76, 88), who plated lar to those which have been demonstrated in out the virus on monolayers of disaggregated chick microorganisms by Yates and Pardee (99) among embryo cells and were able to observe foci, others. The importance of these mechanisms in recognized by the development of rapidly dividing relation to cancer is that they may be involved in cells of altered morphology, which tended to pile the normal control of growth. up on top of one another in distinction from the normal embryonic cells which formed a monolayer. THE INDUCTION OF MALIGNANCY This kind of investigation opened the way to IN CULTURED CELLS quantitative studies of tumor viruses. Whereas established cell strains do not neces Current interest in this field has been greatly sarily give rise to tumors on reinoculation, there stimulated by the observation of Stewart and are many well authenticated instances of primari Eddy (85, 86) that a virus extracted from an in ly nonmalignant strains developing the capacity fectious mouse tumor can produce a great variety to initiate tumors at some time in their history. of tumors when re-inoculated into the animal and The opposite has also been observed. The best can also produce necrotic lesions in cultured mouse documented examples are described by Gey et al. cells. The SE polyoma virus, as it is called, has (33), Sanford et al. (77, 78), and De Bruyn (15). several interesting properties. Thus, it can infect In Gey's laboratory the 14P cell strain was grown related species including the rabbit, rat, and from a culture of normal rat fibroblasts and was hamster as well as the mouse. Also in some cases, repeatedly tested by re-inoculation into rats of after the tumor has been initiated, the virus can the strain from which it was originally derived. It no longer be detected. Finally, the virus itself is produced no tumors for about 2@years. One of the strongly antigenic, and antibodies to it may de sublines then began to produce a rather high mci velop in infected animals. dence of tumors. After a further 10 years of culti These observations suggest that the onset of vation, however, this capacity was lost again. malignancy during the prolonged cultivation of

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1962 American Association for Cancer Research. 486 Cancer R,ese.ardz Vol. 22, May 1962 cell strains may frequently be due to accidental anaerobiosis although the enzyme pattern of the infection with a virus. Perhaps this interpretation cells changed. should be accepted with caution, however, in Radiw@ion.â€”Treatment of cell cultures with view of the loss of malignancy which can also oc x-rays produces multiple chromosomal abnormali cur during cultivation. ties (8). Some cells fail to divide and eventually There have been reports of the passage of other die. The survivors show many kinds of abnormal tumor viruses through tissue culture. In particu ity, including giant cell formation, bizarre mitoses, lar, Coman (12) reported passage of the Shope and unusual morphology. To the author's know! papilloma virus through rabbit skin, and Las edge no attempt has been made to test such cul fargue@ et al. (45) reported passage of the Bittner tures for malignancy by animal inoculation. virus through cultures of mouse mammary tissue. Chev2ical oarei,wgens.â€”Most chemical carcino INTERACTIONS BETWEEN NORMAL gens, though not all, are substances which are AND CANCER CELLS known to increase the incidence of mutation& It was pointed out earlier that cells from ex Earle and Voegtlin's (24) carefully conducted plants of different tissues intermingle indiscrimi studies on the effect of chemical carcinogens on nately. Leighton (47) has performed similar ex cultured cells revealed that treatment of subcu periments in cellulose sponges which provide a taneous fibroblasts with 20-methyleholanthrene three-dimensional matrix and has observed the resulted in gross abnormalities of a kind frequently same behavior. Similar results were obtained when observed in tumors. However, these workers the cells were inoculated into portions of urn systematically studied the transplantability of bilical cord, which might be regarded as a more cells after different periods of treatment with 20- normal kind of matrix (50). This invasive behavior methyicholanthrene and found that after an of normal tissues is unexplained, and it contrasts initial increase it progressively diminished until with Moscona's (60-62) observations that, when after about a year it was very low indeed. During cells from embryonic tissues have been disaggre this time the morphological abnormalities within gated by trypsin, they can reaggregate and segre the cultures continued and even increased. gate into organized structures. In an extension of A related study was carried out by Lasnitzki his experiments Moscona (61) found that S-91 (46), who observed hypertrophy and a greatly in mouse melanoma cells similarly tended at first to creased niitotic rate in organ cultures of mouse segregate from other kinds of cells with which prostate tissue treated with 20-methyicholan they were mixed in reaggregates. However when threne (Fig. 3). However, Sauerteig (80) repeated the aggregates were cultured for some days longer the experiments and attempted to transplant the the melanoma cells infiltrated normal cells in the altered cultures into suitable hosts but was un neighborhood (Fig. 4). Moscona observed that a able to produce tumors by this means. The ma mucinous material (which he calls ECM) was nec lignant nature of the changes must therefore re essary for reaggregation, and that, after cultiva main in some doubt, although they very closely tion in a disorganized manner for some days, cells resembled precancerous changes seen in the lost the capacity to reaggregate. It is tempting to animal. Lasnitzki (47) has also shown that treat speculate that material of this kind may be neces ment of lung organ cultures with 3,4-benzpyrene sary for tissue organization and may be related to the organ-specific antigens which have been found produces changes which might be described as to be absent in cancers (67). precancerous. Experiments carried out by Wolff (98) provide Metabolic stress.â€”Goldblatt and Cameron (34), further information about the interaction be in a classic experiment, attempted to test War tween normal and tumor tissues. He observed burg's theory of carcinogenesis by exposing cul that fragments of normal mouse embryonic tissues tures to intermittent anaerobiosis during 2@ invaded chick tissues when grown in contact with years' continuous cultivation. Morphological ab them in organ culture but did not destroy them. normalities appeared in the treated cultures, and In contrast, when the 5-180 sarcoma was cultured on inoculation into animals some tumors were pro in contact with organ cultures of chick tissues the duced. The significance of these observations is sarcoma cells invaded and destroyed the frag now less certain than it appeared to be at the time, ments. in the light of more recent facts. Adebonojo et al. These experiments illuminate another interest (3) were unable to demonstrate increased ma ing relationship between certain tumor cells and lignancy by treating the L cell with intermittent normal cells. Santesson (79) observed that many

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1962 American Association for Cancer Research. PAULâ€”Cancer Cell in Vitro 437 mouse epithelial tumors grew better in the pres CONCLUSIONS CONCERNING THE ence of connective tissue. Schleich (82) also NATURE OF THE CANCER showed that the Yoshida ascites sarcoma of the rat PROCESS would only grow satisfactorily in vitro in the pres Cancer always involves a local breakdown of the ence of fibroblasts. De Bruyn (15) demonstrated tissue homeostatic mechanisms. Consequently, a similar relationship in connection with her MB cancer cells behave rather like tissue culture cells; strains; and finally Wolff (98), in the experiments and similarities between them, far from implying referred to above, observed that although the that all cultured cells become malignant, serve to 5-180 sarcoma was very difficult to grow in culture emphasize that cancer cells achieve almost corn by itself in his conditions nevertheless it grew very plete autonomy and become, in effect, tissue cul well in contact with fragments of normal organs tures in i,it'o. When this proposition is accepted it and could be passaged in this manner through becomes clear that some of the phenomena which many subcultures. occur readily in cultured cells can probably be ex eluded as being specific for the cancer process. ONCOLYTIC AGENTS Thus, many morphological and metabolic ab Since the rational approach to cancer therapy normalities and the emergence of chromosomal has so far proved unproductive, there have been variations are more likely to be results of the can many purely empirical attempts to evolve thera cer process than etiological factors. peutic agents specific for cancer cells, and cultured The fundamental lesion involved in the break cells have been used as test objects. In some down of the homeostatic mechanisms is not un studies, such as those of Biesele (9, 10) and derstood. It may involve the surface properties of Eichorn et al. (25), an attempt was made to screen cells (particularly their mutual adhesiveness), the a large series of metabolic analogs for their toxicity production of cell-specific antibodies (35), or against lines of cancer cells on the one hand and again some kind of feedback control of cell growth normal cells on the other. On the whole, little dif and migration operating through metabolic path ferential toxicity was found. Eagle and Foley ways. Probably multiple mechanisms are involved, (20, 21) conclude that these agents exhibit a gen and each of these may be controlled by one or era! cytotoxicity for all kinds of growing cells and several genes. The â€œdeletionâ€•theory (73) proposes that their value as oncolytic agents is simply a that cancer is a consequence of successive somatic measure of their cytotoxicity. This conclusion is mutations which result in the loss of all these in accord with the proposition that there is no mechanisms and permit the cell to become inde general qualitative difference between the metabo pendent of local control. It may not be necessary lism of cancer and normal cells. to postulate successive multiple mutations. Some Oncolytic viruses.â€”Certain viruses have been of the control mechanisms may be relatively found to have some specificity for certain cancer complicated and may, for instance, consist of cells both in vitro and in vivo. However, it is im several enzymes involved in the synthesis of a practicable to use these as therapeutic agents be specific substance. Each enzyme may be con cause, as Moore (57) points out, the patient de trolled by one or more genes, and consequently a velops viral antibodies more quickly than the single lesion affecting any one of the genes or the virus destroys the tumor. protein-synthesizing systems they control might Tuaor antibodies.â€”Attempts have been made result in a breakdown of the whole system and the to produce antibodies against tumor cells, and establishment of a cancer. A mechanism of this some limited success has been obtained. For in kind would be in accord with current thinking in stance, Prehn and Main (75) found that specific the field of molecular biology and would not con ffict with the known facts. antibodies were produced against methyleholan Much of what is known of the etiology of cancer threne-induced tumors. On the other hand, these would fit a theory which implicates damage to the workers found that antibodies were not produced genetic apparatus of the cell. Thus, a proportion against spontaneous tumors. This is probably a of cancers might arise spontaneously by somatic special case, methyleholanthrene combining with mutation. On the other hand, specific mutagens cellular proteins to form new antigens. In the might not only increase the incidence of random majority of cases where antibodies have been pro mutations but might be expected specifically to duced against tumor cells it has been found that increase certain kinds of mutations. Finally, they are equally effective against some of the viruses might operate by a process analogous to normal cells of the host. transduction. The point of convergence of all

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FIG. 1.â€”Neurites and neuroepithelium in a culture of a FIG. 3.â€”Photomicrographs of sections of mouse prostate metastatic nodule of a sympathicoblastoma in a 1-year-old gland maintained in organ culture. X115. Stained H. & E. child. Seventeen days in vitro. Stained by Bodian's protargol a: Grown for 21 days in normal medium. b: Grown for 11 days method. (Courtesy of Dr. Margaret R. Murray.) with 4 @ig/ml methylcholanthrene, followed by 10 days in Fio. 2.â€”Top: â€œEpithelialâ€•cell or pericyte from a tumor of normal medium. (Courtesy of Dr. I. Lasnitzki.) the ankle in a 27-year-old male. Twenty-four days in culture. FIG. 4.â€”Infiltration of 7-day disaggregated chick cells by Stained by Bodian's protargol method. Bo&nn: Zimmerman's mouse pigmented melanoma cells after 72 hours in culture. At capillary pericyte. Golgi silver impregnation. (Courtesy of Dr. first the two cell-types segregated into separate zones. X275. Margaret R. Murray.) (Courtesy of Dr. A. Moscona.)

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Cancer Res 1962;22:431-440.

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