the nucleo-cytoplasmic ratio and cancer · infusoria may also be clearly inferred from the fact...

THE NUCLEO-CYTOPLASMIC RATIO AND CANCER T. BRAILSFORD ROBERTSON

From the Darling Laboratories of Physiology and Biochewtistry, University of Adelaide, South Australau

It has been believed since 1908 that the process of growth, in animals and in plants, is autocatalyzed; that is, that i t is determined in its magnitude and in its velocity by some underlying chemical process, the “master-reaction” of growth, which is of such a nature that some product of the reaction facilitates its further occurrence (10, 13, 14).

A process of this nature might proceed indefinitely, at an unceasingly augmented velocity, if it were not for the fact that two necessary consequences arise out of its occurrence, the one being the consumption of the substrates which enter into the reaction, and the other the accumulation of its products, including the autocatalyst itself. Either of these events may ultimately bring the master-reaction to a standstill. Exhaustion of any one of the substrates, which are the “raw materials” out of which protoplasm is synthesized, must obviously terminate the process of growth, while, even if this does not occur, the accumulation of the products, as in other chemical reactions, must) progressively augment the velocity of the reverse reaction, until at length it equals the velocity of the forward reaction and a condition of stasis is imposed. An equilibrium thus attained can be altered only by some extraneously imposed change of the conditions, such as an increase of the concentration of available substrates (t)he ‘‘ nutrient-level”), removal of the products of the reaction (for example, mutilation,) or a modifica- tion of the physical conditions, such as temperature.

In the higher animals it is demonstrable that the circumstance which imposes limitations upon the extent of growth, so that a mouse, for example, cannot possibly grow to the size of an elephant, is not exhaustion of the substrates, but accumulation of the products of growth. The nutrient level in the tissue-

292

THE NUCLEO-CYTOPLASMIC RATIO AND CANCER 293

fluids is, as a matter of fact, extraordinarily constant throughout life. It is maintained a t an almost invarying level by a series of balanced dynamic equilibria which are analogous to those which maintain a constant temperature in the homoiothermal animals, or it may be compared to the level of water in a reser- voir, in which the inflow (the acts of feeding and absorption from the intestine) is greatly in excess of the outflow (consumption by the cells) and a constant level is maintained by means of an overflow (the exogenous metabolism). That the nutrient level in the adult is, in fact, sufficient to permit vigorous growth is shown by the fact that mutilation of an adult animal, in which growth has ceased, nevertheless inaugurates repair, and also by the fact that neoplasms, in the very environment (i-e., tissue-fluid) which prohibits growth of the normal tissues, nevertheless display capacity for growth which is only limited by the capacity of the animal to resist invasion of its essential tissues.

It is confinement of the community of cells to a limited enclosed volume of nutrient medium which imposes ultimate limitation upon their capacity to multiply. Cell communities with no enclosed circulatory system, such as the corals, are checked in their multiplication only by gross external limitations, such as lack of oxygen or nutrient,s, or approach to the surface of t8he sea. Tissue cells which are removed from vertebrate animals and cultivated in vitro multiply indefinitely, provided the nutrient m e d i u m i s constantly changed, and may outlive by many multiples the normal life-duration of the animal from which the cells were originally derived (3). Some product of the growth-process, other than the cells themselves, accumulates within any closed volume of nutrient medium and this it must be which primarily determines the limit of growth of all the higher animals.

The autocatalytic character of the process of reproduction in cultures of bacteria has been referred by McKendrick and Pai t o the inherent capacity of each cell to reproduce itself, due to the presence within it of catalyzers capable of effecting proto- plasmic synthesis, cessation of reproduction being ultimately

294 T. BRAILSFORD ROBERTSON

compelled by the exhaustion of the substrates, i.e., of the available foodstuffs (9). We have seen that this limiting factor is not normally operative in vertebrate animals and that it is not the sole factor which determines cessation of reproduction in unicellular communities which are confined to a limited volume of nutrient medium may be shown by the fact that mere dilution of a densely populated medium, in which reproduction of infusoria has ceased, by the addition of (‘ buffered” distilled water, which cannot enhance but on the contrary must diminish the “nutrient level,” suffices t o partially restore the capacity of the medium to support reproduction (17).

When two infusoria are isolated together into the same small drop of culture-medium, the number of individuals produced after twenty-four hours is not twice but several times or even, it may be, as much as sixteen times as great as the number of individuals produced by a single infusorian isolated into an equal volume of medium (16). This mutually accelerative or ‘‘ allelocatalytic effect” of contiguous cells is not due to conjugation because, under the conditions of the experiment, conjugation does not occur. It is not due to contamination of the medium by an extra “charge” of associated bacteria which accompany the second cell during its introduction into the medium, for allelocatalytic effect is demonstrable ill media thickly inhabited by bacteria, in which the additional infection introduced with the infusoria would contribute a negligible proportion of the total bacterial population. It must be attributed to the shedding into the medium, by the cells which it contains, of a substance which promotes reproduction. When an infusorian (Enchelys) has been isolated into fresh hay infusion, or into distilled water to which a suitable reaction and tonicity have been communicated by alkaline salts, the presence of an accelerative agent in the medium can be demonstrated, directly after the first cell-division, by isolating one of the daughter-cells into a fresh sample of the medium. The reproductive rgte of this re-isolated cell is always much less than that of the cell which has been left undisturbed in its surrounding medium. The presence of such a substance in a medium inhabited by


infusoria may also be clearly inferred from the fact that the reproductive rate of isolated infusoria is increased by mere reduction of the volume of the culture-medium, 50 that the number produced from a single individual after 24 to 48 hours varies almost in inverse proportion with the volume of the medium into which it is isolated. If the volume is too great, reproduction may fail altogether to occur (17).

Finally, glass-distilled water which has been rendered faintly

alkaline by the addition of 1 cc. of - Na2C03 per 100 cc., and

which has been inhabited by multiplying infusoria for 48 hours, may be shown to contain an agent which accelerates the reproduction of infusoria in the following manner: The water is freed from infusoria by heating to 50” C. to immobilize them, followed by filtration through a double filter paper. The filtrate is then neutralized to cochineal indicator by the addition of n/100 hydrochloric acid and evaporated on a boiling water- bath to one half its original volume. A small precipitate of coagulated protein is removed by filtration and the fluid, stored in flasks plugged with cotton, is boiled several times a t intervals of two or three days to sterilize it. This fluid, when added to “buffered” distilled water or to hay infusion, greatly accelerates the reproductive rate of infusoria isolated into these media. No comparable acceleration of reproductive rate could be brought about by alteration of the p H of the media (17).

I t has been shown that the auto-acceleration of reproduction in a cellular community and the ultimate dimensions or population attainable by the community are alike determined by the accumulation of a product in the medium which is derived from the cells which inhabit it. The autocatalytic character of the growth-process permits us to assume that one and the same product may be responsible for the initial acceleration and the final retardation, just as, in an autocatalytic reaction in labora- tory glassware, the accumulation of the products of the reaction, including the autocatalyst itself, ultimately brings the forward reaction to a standstill. I n the first half or autokinetic phase of the autocatalytic curve of growth, increase of the autocatalyst

N 10


level in the medium must lead to increase of the reproductive rate, while in the second half, or autostatic phase, increase of the autocatalyst level in the medium must lead to decrease of the reproductive rate.

The source of the autocatalyst of cellular reproduction is the nucleus. This may be inferred from the fact that the growth of the nucleus, after each nuclear division, is a t first slow and later much more rapid, while the growth of cytoplasm, on the contrary, is comparatively uniform in rate (12). The nucleus itself, therefore, elaborates a catalyzer which accelerates its growth, while the growth of cyt>oplasm reveals no such auto- acceleration. The autocatalyst, however, is retained within the nucleus until the dissolution of the nuclear membrane during nuclear division permits it to issue into the external medium, In the inter-divisional period, in fact, the cell is insusceptible to accelerative agencies in the nutrient medium; they cannot penetrate the nucleus until nuclear division occurs. Thus, if two infusoria (E’nchelys) are isolated together into the same sniall drop of hay infusion, tlhey do not begin to divide any earlier than a single individual isolated into a drop of similar size. But once division has occurred, acceleration begins to manifest itself. Similarly the “ lag-periods ” of single infusoria isolated into varying volumes of culture fluid do not appreciably differ, but once cell division has occurred, then the intervals between successive divisions are much abbreviated in the smaller volumes of culture fluid, and multiplication is correspondingly accelerated. The accelerative agent which is discharged by multiplying infusoria into “buffered ” distilled water does not abbreviate the ‘‘ lag-period ” of infusoria isolated into nutrient media which contain it. Yeast extract shortens the “lag- period ” only to a relatively insignificant degree while, subse- quently to the first division, the reproductive rate is decidedly enhanced (17).

We must infer from these observations that a cell recently isolated into a culture medium is relatively insusceptible, during the period preceding the first division, to agents which accelerate nuclear synthesis. For the time being the nucleus is closed to


outside influences and carries with it the characteristics of the nuclei of the cells which inhabited the parent culture. If the parent culture is old and densely populated, reproduction has almost ceased in it, and correspondingly we find that the lag- period is immensely prolonged. If, on the contrary, the parent culture is a youthful one in which a high reproductive rate is still maintained, then the lag-period is brief (11, 15).

During the periods between nuclear divisions, therefore, each nucleus retains the charge of autocatalyst with which it was provided at the conclusion of the preceding division and adds to it in the course of the nuclear synthesis which is rendered possible by its presence. The auto-acceleration of nuclear growth is attributable to this increase of the included autocatalyst. At the next division the autocatalyst is shared between the nuclear materials and the surrounding medium in a proportion determined in part by its relative solubility in the two media, and in part by its affinity for chemical substances within the nucleus. A t the end of this redistribution the autocatalyst is partitioned between the external medium, on the one hand, and on the other hand the nuclear substances with which it combines or in which it is dissolved. The nuclear membrane is then re-formed, and the autocatalyst within the nucleus is again shut off from dispersal into the surrounding medium until the occurrence of the next succeeding division. At any moment the concentration of autocatalyst within the nucleus will therefore be represented by

L. + Xend., where Xex. is the concentration of autocatalyst which was left in the nucleus a t the conclusion of the last division and Xcnd. is the concentration of autocatalyst which has accurriulated within the nucleus since that event occurred. If the sum of these two fractions of the autocatalyst-content of the nucleus exceeds the concentration which marks the conclusion of the autokinetic phase of the curve of autocatalysis, all further accumulation of nuclear autocatalyst will tend to progressively delay nuclear synthesis and, in consequence, if the initisl charge represented


by Xcx, is very large, owing to excessive accumulation of the autocatalyst in the nutrient medium, then a relatively small addition of Xorld. may result in such retardation of the rate of nuclear synthesis that it can never gain sufficiently upon the rate of cytoplasmic synthesis as to permit the attainment of the nucleo-cytoplasmic ratio which is requisite for cell division (18). The sequence of events within a cell inhabiting a medium which has for long been densely populated may be illustrated by the accompanying diagram in which nuclear and cytoplasmic growth in cells arising in young and old communities, respec- tively, are diagrammatically compared.

FIG. 1. Diagrairinitttic reprt?scnt,ation of the relative chrmgeP in nuclear t~ricl cytoplasmic volume during thr growth of :L cell (1) originating in a young coniniunity and (11) originntirig in RII old caniinunity. The volnrnes are expressed in units so adjueted thxt thc ratio necessary for division is unity. In the cell arising in LL young conirnunit,y this ratio its ntt,rrinotl during, or soon after, the autokinetic phase of nuclenr growth. I n the cell arising in an old commuiiit,y the inclusion of autocatdyst, ticrived from the nntricnt Incltlium at thc preceding division, mity inhibit miclear growth ticfore the nitio rcqiiisito for divisian is atiaincd.

A comparison of these diagrams at once reveals two possi- bilities. The one is that a diminution of the nucleo-cytoplasmic ratio requisite for division might enable the type of cell inhabiting the old and densely populated medium to divide, and its daughter-cells, if they inherited like capacity to divide at a low nucleo-cytoplasmic ratio, would also he enabled to divide until a t last the further accumulation of autocatalyst in the medium consequent upon these divisions sets a limit to their multiplication also, when failing the appearance of cells able to divide a t still lower ratios, developmental stasis must occur. The second possibility is that the nucleo-cytoplasmic ratio finally attained


in a cell which has not recently undergone division may be far smaller than the normal characteristic ratio at division. In seeking to define a cell by its nucleo-cytoplasmic ratio, therefore, we must also be careful to define its physiological age, i.e., the time which has elapsed since the division from which it originated,

Each of these inferences is confirmed by well-known observations. Thus Conklin has shown (2) that the original sup- position of Hertwig ( 7 ) that the nucleo-cytoplasmic ratio at the moment of cell-division is a constant, peculiar to each species, is true only if the measurements are confined to particular tissues or, in the earlier stages of development, to particular blastomeres. As development progresses, the nucleo-cytoplasmic ratio a t the moment of cell-division diminishes, so that the increase of nuclear material with development bears a diminish- ing ratio to the number of cells produced.

On the other hand, Macallum has shown that the number of cells in the human sartorius muscle does not increase after the embryo has attained a length of 170 mm. (8), the whole of the subsequent growth of this muscle being due to increase of size of the cells, chiefly, we may presume, their cytoplasm. The fall of the nucleo-cytoplasmic ratio, in adult as compared with embryonic tissues, thus arises in two ways: in the first place in consequence of a real diminution of the nucleo-cytoplasmic ratio at the moment of cell-division and in the second place in consequence of the length of time which has elapsed, in the case of adult cells, since the last cell-division occurred, during which time nuclear growth has been greatly retarded, while cytoplasmic growth has been enabled to continue without interruption by cell-division.

The progressive step-by-step diminution of nucleo-cytoplasmic ratios a t the moment of cell-division which accompanies the development of a niulticellular organism involves two consequences. It enables the cell-community to evade the consequence of autocatalyst-accumulstion in the enclosed nutrient medium, and thus to continue multiplication at a n autocatalyst level which would otherwise impose developmental stasis upon the community. In the second place it results in the production

300 T. BRAILSFORD ROBERTSON I

of a variety of cell-types of differing physiological behavior, which react in differing and characteristic fashions to theenviron- ment and thus produce the variety of cytoplasmic structures which collectively constitute the diverse tissues of a higher animal or plant,

Howover long developmental stasis may he deferred by this device, however, i t must ultimately happen that no further reduction of nucleo-cytoplasmic ratios is possible. The genetic constitution of the parent germ cells, from which the community arises, permits a graduated series of such diminutions and when the end of the series is reached, the cessation of development is not far off. This, then, is what ushers in the attainment of the adult condition.

Let this condition of stasis be interrupted by removal of a portion of the products of the growth-process, as, for example, by mutilation. Repair is inaugurated and obviously, among the various types of cells available to replace the missing tissues, t>hose which are ontogenetically the most recent and which have the lowest nucleo-cytoplasmic ratios requisite for division will be the best adapted to the existing high autocatalyst level and will multiply most rapidly. Let, the restored tissue be again removed and again the process of repair by cells of predominat- ingly low nucleo-cytoplasmic ratios will occur. If this happens again and again, it will obviously be equivalent to a process of selective breeding, and if the genetic constitution of the cells surrounding the injured locality permits, even very seldom, the production of a cell with an abnormally low nucleo-cytoplasmic ratio at division, this type of cell may hypothetically find itself able, not only to replace the missing tissue, but to continue multiplying after this has been accomplished, to invade other tissues, and to overwhelm them by superior reproductive capacity and prior appropriation of nutrients. Here, then, we have a possible condition of origin of cancer cells.

Such excessively low nucleo-cytoplasmic ratios at division might arise from a peculiarity in the genetic constitution of certain cells in the locality in which the neoplasm originates, or they might originate in a tendency to asymmetric mitosis,


producing daughter-cells with unequal nuclei, provided that the daughter-cell with the smaller nucleus retained the capacity to divide again upon reattainment of the ratio which it exhibited at the moment of its production.

The occurrence of asymmetric mitoses in cancer has been recognieed ever since v. Hansemann drew attention to them (4,5, S), but it has generally been assumed, without any evidence to that effect, that it is the cell possessing the larger nucleo- cytoplasmic ratio which contributes the malignant type of tissue. This belief originates in an over-emphasis of the superficial resemblance of cancer tissue to embryonic tissue, a resemblance which, after all, may extend no further than this: that both types of tissue are rapidly proliferating, with all the physiological and metabolic consequences which this fact entails. Ontogenetically the two tissues represent opposite poles of development and since the cancer cell shows, by its high reproductive capacity, a high degree of adaptation to the cellular environment of an aged animal, to that extent at least it differs very greatly from an embryonic cell. If the above is a true account of the origin of cancer, then, in the asymmetric mitoses which occur in neoplasms, the malignant cell must be the one possessed of the smaller nucleo-cytoplasmic ratio, the other must be doomed to sterility and ultimate extinction by its successful competitors.

The above hypothesis as to the origin of cancer was first put forward by the author in his book on the Chemical Basis of Growth and Senescence (18). While this book was in the press, the paper of Sokoloff appeared in which numerous measurements of nucleo-cytoplasmic ratios in various types of cancer are reported and compared with the ratios found in adult and embryonic tissues (19). He thus summarizes his main conclusions: “In the tissues of malignant tumors two types of cells are present; one, in which the nucleus is much enlarged, possesses the character of a depressed cell; the other, with nuclei not especially enlarged, exhibits no evidence of any such depression. Cells of the latter type, that is, cells showing no evidence of depression, whether in mouse carcinoma or in

20


various types of human carcinoma, have a nucleo-cytoplasmic ratio approaching that of embryonal cells.”

The latter of these conclusions appears at first sight to stand in absolute contradiction to the inferences outlined above, but upon closer analysis of the actual measurements obtained by Sokoloff the contrary is found to be the case. In fact, his figures fully substantiate the conclusion that in the asymmetric mitoses in cancer the cell with the larger nucleo-cytoplasmic ratios is sacrificed because it is unable to multiply and in consequence is overwhelmed by the enveloping cells of the type possessing the lower nucleo-cytoplasmic ratios. As regards the comparison with embryonic cells, it has not in fact been made, for, as we must again recall, and as Conklin and many others have shown by actual measurement, the nucleo-cytoplasmic ratio differs with the physiological age of the cell. Cancer cells are physiologically young, that is, recently produced. They have had no time to accumulate their full complement of cytoplasm. For this reason they must inevitably appear to have high nucleo-cytoplasmic ratios when compared with adult cells, in which nuclear growth ceased long before cytoplasmic growth had reached its limit. The comparison, to be accurate, should be instituted between the nucleo-cytoplasmic ratios of cancer cells at the moment of division and the tissue cells of the host at the moment of their production. In the single instance of which Sokoloff gives an illustration of a “very young cell,” the nucleus, as he is careful to point out, is very small.

On the other hand, the cells with exceptionally large nuclei &re all found to be undergoing degeneration, and they are likewise much inferior in number to the cells having smaller nucleo- cytoplasmic ratios. Thus he states ‘ I There are some sections in which every eighth or tenth cell shows this type of greatly enlarged nucleus” and later ‘ I . . . cells showing those nuclear changes to which the terms depressed or degenerated have been applied are dying cells, and they are not those which play any part in the growth activity of the malignant tumor.” It follows, of course, that it is the cells possessed of lower nucleo- aytoplasmic ratios which constitute the malignant tissue.


One consequence of our hypothesis is therefore experimentally verified. It is, in fact, the cell of lower nucleo-cytoplasmic ratio possibly produced in the asymmetrical mitoses in cancer which is the malignant type of cell. The other consequence of our hypothesis, that the nucleo-cytoplasmic ratio at the moment of division must be less than the nucleo-cytoplasmic ratio of normal adult tissue at the moment of its production, has not as yet been experimentally tested. It appears that only direct measurement of the nuclei of the two types of cells at the moment of actual division will enable us to test this deduction. Unfortunately, as le Breton and Schaeffer have recently pointed out (l), the determination of nuclear volume is not an altogether reliable measure of the mass of distinctively nuclear components (nucleic acids), because, as Conklin and others have shown (2), the nucleus contains other constituents besides chromatin and some of these may even be of extra-nuclear origin. If the proportion of the various constituents to one another should vary from one tissue to another, as we know it varies with the physiological age of the cell, then volume would be no longer a measure of distinctively nuclear mass. On the other hand, the ingenious chemical method of estimating the nucleo-cytoplasmic ratio devised by le Breton and Schaeffer is at present only applicable to masses of tissue and not to individual cells at the moment of their production by division. Probably the nearest approximation to a solution of this problem which is at present attainable must be sought in direct measurements of nuclear and cytoplasmic volume at the moment of division, assuming that the relative proportion of the several nuclear constituents is always the same at corresponding stages of the nuclear cycle. This assumption, to which the observations of Conklin (2) lend some measure of support, has nevertheless not yet been shown to be invariably correct, even when applied to the nuclei which arise from normal mitoses, and its applicability to nuclei which arise from abnormal mitoses is, of course, a matter of even greater uncertainty .

304 T. BRAILSFORD ROBERTBON

SUMMARY

The measurements, by Sokoloff, of the nucleo-cytoplasmic ratio in cancer cells are in harmony with the view that cancer arises from the production of cells which are capable of undergoing division at abnormally low values of the nucleo-cytoplasmic ratio. In particular, they demonstrate that when such cells arise by asymmetrical mitoses, the daughter-cell with the larger nucleo-cytoplasmic ratio is unable to multiply and is ultimately overwhelmed by the multiplication of the malignant cells, arising from the daughter-cell which possesses the lower nucleo- cytoplasmic ratio.

REFERENCES (1) BRETON, E. LID, AND BCHAIUFFER, G.: Travaux de I'Institut de Physiol., Faculte

(2) CONKLIN, E. G.: J. Exper. Zool., 1912, xii, 1. (3) EBPJLINQ, A. H.: J. Exper. Med., 1910, xxx, 531. (4) HANEEMANN, B.: Virchows Arch. f. path. Anat., 1890, cXix, 289. (5) HNVSIOMANN, B.: Vimhows Arch. f. path. Anat., 1891, clwiii, 350. (0) HANSHIMA", B.: Virchows Arch, f. path. Anat., 1802, c d x , 430. (7) HERTWIQ, R.: Monohen. med. Wchnschr., 1903. (8) MACALLUM, J. B.: Bull. Johns Hopkins Hosp., 1898, ix, 208. (9) MCKENDIUCK, A. G,, AND PAI, M. K.: Roc. Roy. SOC. Edinburgh, 1911, xxxi,

(10) OSTWALD, Wo.: Vortrllge und Aufslltze ueber Entwicklungamech. herausgeg.

(11) P B ~ O L D , W. J.: J. Hygiene, 1914, xiv, 215. (12) POPOFF, M.: Arch. f. Zellforsch., 1909, 111. (13) ROBERTSON, T. BRAILEFORD: Arch. f. Entwicklungemech. der Organismen,

1908, xxvi, 108. (14) ROBERTSON, T. BRAILSFORD: Arch, f. Entwicklungsmech. der Organismen,

1909, h i , 29. (15) ROBERTSON, T, BRAILSFORD: Biochem. Journ., 1921, xv, 595. (10) ROBIRTSON, T. BRAILSFORD: Biochem. Journ., xv, 612. (17) ROBHIRTSON, T. BRAILSFORD: J. Phyeiol., 1922, lvi, 404. (18) ROBERTEON, T. BULBFORD: "The Chemioal Basis of Growth and Senescence."

(19) SOKOLOFP, B.: J. Cancer Res., 1922, vii, 305.

de Medicine de Strasbourg, Paris, 1923.

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v. Wilh. Roux, Heft 5, 1908.

Philadelphia and New York, 1923.

the nucleo-cytoplasmic ratio and cancer · infusoria may also be clearly inferred from the fact...

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