Inhibition of Cell Division: A Critical and Experimental Analysis1

Inhibition of Cell Division: A Critical and Experimental Analysis' SEYMOURGELFANT Department of Zoology, Syracuse Uniz'ersity,Syracuse, New York

I Introduction It Analysis of the Inhibition of Cell Division III Experimental Systems Used A. Mouse Ear Epidermis m Vitro B. Mouse Body Skin Epidermis m Vrzm C. Rat Uterus rn Vtvo I V Synopsis of Inhibitors Used A. Colchicine B. Podophyllin C. Vincaleukoblastine (VLB) D. Chloral hydrat: E. Mercaptoethanol F. Malcuric Acid G Nitrogen Mustard H Aminopterin 1. Actidione V. Effects of Various Inhibitors on Mitosis im Vitro A Mctaphase Inhibitors Compared with Colchicine B Energy Metabolism Inhibitors C. Hormones VI Effects of Various Inhibitors on Mitosis in Vrvo A Mouse Body Skin Epidermis B Rat Uterus O VII Effects of Various Inhibitors on D N A Synthesis i n V I ~ Vand in V ~ t o A Mouse Ear Epidermis In Vitro B Mouse Body Skin Epidermis m Vtvo VIII Effects of Various Inhibitors on Growth and on Mitosis m VI vo IX Comment on the Inhibition of Cell Division by X-Irradiation X Comment on Cancer Chtmotherapy Screening Methods XI Concluding Remarks References

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I. Introduction In this review, inhibition and progress of cell division were viewed as complementary aspects of the same problem. It was assumed that all of the biochemical and physiological events involved in the process of cell division can 1 This investigation was supported (in part) by a research grant RG-7485 (C4) from the Nationaf Institutes of Health, United States Public Health Service.

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SEYMOUR GELFANT

be dissociated from one another by selective experimental inhibition. The question of the inhibition of cell division was therefore approached by focusing on the process of cell division. 11. Analysis of the Inhibition of Cell Division

Figure 1 presents a visual and synoptic analysis of the major events of cell division as seen from several points of view; physiological, morphological, biochemical, and temporal. The ideas and the information expressed in Fig. 1 were derived from the following sources: Mazia (1956, 1959, 1960, 1961a,b). Swann (1957, 1958), Ris (1955, 1957, 1961), Stern (1956, 1959, 1960a,b), Wilson and Morrison (1958), Wilson (1959), Brachet (1957), Bajec (1957, 1958, 1959), Ostergren et al. (1960), Makino and Nakanishi (1955), Jacobson and Webb (1952), Boss (1955), Lima-De-Faria (1958), Lettri and Lettre (1959), Mitchison (1957, 1958, 1961), Zeuthen (1946, 1953, 1958, 1961), Scherbaum (196oa,b), Prescott (1961), Wolpert (1960), Gross and Spindel (1960), Howard and Pelc (1953), Lajtha (1957), Gelfant and Clemmons (1955), Gelfant et al. (1955), and Gelfant (1960c, 1962). W e are now in a position to discover what is meant by the inhibition of cell division. The analysis that follows is based upon three themes: continuity, dissociability, and dependence or independence of individual or groups of events. For example, by focusing on the behavior of the chromosomes, one can establish three major physiological events that occur during cell division : chromosome reproduction, chromosome movement, and cytoplasmic cleavage. If chromosome reproduction is inhibited, chromosome movement and cytoplasmic cleavage will not take place. If chromosome movement is inhibited, the third event will not occur, and if cytoplasmic cleavage is blocked, the end result of cell division, the formation of two daughter cells will not be achieved. Thus all three events normally occur with strict continuity, are interdependent, and can be dissociated from one another by appropriate inhibitors. If one focuses instead on the subsidiary events that occur during interphase and mitosis as shown in Fig. 1, the situation is much more complex. The theoretical implications of continuity and of dissociability still apply, but the interdependence of the subsidiary events upon one another may not be as crucial as it is with the major categories within which they are listed. Reproduction of the mitotic centers in sea urchin eggs, for example, can be blocked without affecting D N A synthesis (Bucher and Mazia, 1960); or each of the two anaphase movements in grasshopper spermatocytes, chromosome to pole or spindle elongation, can be specifically inhibited without affecting the other (Ris, 1949; Nakahara, 1952). Nevertheless, all of the events in any one category are related functionally, and also in the sense that inhibition of any one of them will prevent the cell from entering the next major category or stage of cell division. Thus it can be shown that a cell will not enter mitosis if any of the interphase events are inhibited;

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3

such as energy production [in sea urchin eggs, Tetvuhynzenu, or excised pea roots (Swann, 1953; Krahl, 1950; Zeuthen, 1958; Amoore, 1961a,b)}, sulfhydryl conversions [in lily anthers and algae (Stern, 1960b; Hase et al., 1960)], or DNA synthesis [in a wide variety of cell types (Biesele, 1958)l. Damage to the nucleolus in early prophase [grasshopper neuroblasts (Gaulden and Perry, 1958; Gaulden, 1960)} or prevention of nuclear membrane breakdown during prophase [excised pea roots (Hadder and Wilson, 1958)] stops the cell from entering metaphase. If the spindle is disoriented during metaphase [wide variety of cell types (Eigsti and Dustin, 1955)) anaphase will not occur, and if anaphase movement is inhibited, telophase will not take place [grasshopper spermatocytes (Ris, 1949; Nakahara, 1952) 1. Finally, if cytoplasmic cleavage is blocked in telophase [sea urchin eggs, amoeba (Marsland, 1956; Prescott, 1961)} the two daughter cells are not formed. To complete the cycle, one can also prevent the occurrence of an interphase event by a previous influence during mitosis. This has been demonstrated in sea urchin eggs (Bucher and Mazia, 1960), for when the chromosomes are maintained in the condensed state and are prevented from uncoiling, DNA synthesis does not occur even though the necessary percursors are present. Superimposed upon the dependent interrelationship of events from category to category is the question of independent continuity of events both on a subsidiary and on a precursor level. These events may go through their own continuous cycle irrespective of other closely related or distantly removed events, and these too can be dissociated experimentally. The first example to illustrate this point involves the synthesis of DNA and of DNA precursors. The observation that DNA synthesis occurs even though mitotic center reproduction is inhibited [in sea urchin eggs (Bucher and Matia, 1960) 1 has already been alluded to. To go one step further, one can dissociate the formation of macromolecular D N A from the independent synthesis of its precursors. A metaphase block in sea urchin eggs prevents the formation of macromolecular DNA, but it does not prevent the cyclic accumulation of DNA precursors during the block (Bucher and Mazia, 1960). This observation is supported by results in Eschevichia coli (Kanatir and Errera, 1954) where acid soluble D N A precursors (phosphorus, purines, pyrimidines, and pentoses) accumulate even though D N A synthesis is inhibited. Another dissociable biochemical event closely related to D N A involves the independent synthesis of non-histone chromosomal protein [in epithelial cells of the rat uterus (Gelfant and Clemmons, 1955)], where increased nuclear volume and non-histone protein content occurs in the absence of D N A synthesis. The reciprocal observation has also been reported [in bacteria and in onion root tips (Maruyama and Lark, 1959; Krause and Plaut, 19601, where the periodicity of DNA synthesis continues even though protein synthesis is inhibited. And a natural example of D N A and non-histone protein dissociation can be found during spermiogenesis (Ris, 1959). In this case the non-histone

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FIG. 1. Diagrammatic representation of the physiological, morphological, and biochemical events that occur in preparation for and during each stage of cell division. Upper right. Temporal scheme of the cell division cycle.

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SEYMOUR GELFANT

protein disappears from the spermatid nucleus leaving the most elementary microfibril, 40 A. thick, composed of DNA-protamine macromolecules. The last illustration involving the independent behavior of D N A or of its precursors can be drawn from experiments on temperature induced synchronization of growth in Tetrahymena (Iverson and Giese, 1957; Scherbaum et al., 1959; Scherbaum, I960a), where macromolecular DNA, deoxyribosides, and deoxyribotides accumulate, and thymidine is incorporated into DNA even though cell division is being blocked during the heat treatment. Morphologically this increased accumulation of DNA appears to be associated with macronuclear volume increase (Williams and Scherbaum, 1959; Holz, 1960). The second example of an independent, continuous, and dissociable biochemical event involves the cyclic increase in respiration that occurs during interphase. This can be demonstrated in sea urchin eggs (Zeuthen, 1951, 1953), where the normal rhythmic increase in oxygen consumption persists even though cell division is blocked at metaphase. The independence of energy yielding respiration can also be demonstrated by its being dissociated from D N A synthesis. In bone marrow cells, for example (Lowrance and Carter, 1950), DNA synthesis can be inhibited without affecting the increase in respiration that normally occurs. Also in yeast cells (Katchman, et al., 1959) inhibition of DNA synthesis does not prevent the metabolism of phosphorus associated with the build-up of high energy phosphate compounds. Finally, there are a few additional examples of independent dissociable subsidiary and precursor events related to chromosome movement and to cytoplasmic cleavage. The independent movement of each chromosome during anaphase can be demonstrated by selectively damaging individual chromosomal fibers [primary spermatocytes of the German crane fly (Forer, ca. 1962)]. The actual process of reproduction of the mitotic centers in echinoderm eggs can be dissociated from the splitting and separation of the centers (Mazia, et al., 1960). Inhibition of cell center duplication does not prevent the separation and migration of the pre-existing duplex centers because each one of them can be realized as an actual pole in the four-polar division that occurs when the block is removed. Also the synthesis of the astral and spindle precursor components can be dissociated from the organization of these structures in sea urchin eggs (Kawamura and Dan, 1958). In this case the cyclic accumulation of astral and spindle precursor sulfhydryl-containing proteins continues even though morphological formation of the asters has been suppressed. The last example focuses on the independence of events involved in cleavage in sea urchin eggs. To begin with, the cleavage furrow can be initiated even prior to prophase - before any sign of nuclear events (Zimmerman and Marsland, 1960). Secondly, the submicroscopic structural changes in the cell cortex can be dissociated from those occurring in the mitotic apparatus (Monroy and Montalenti, 1947) because the spindle can be disoriented in metaphase without affecting the cyclic variations in birefringence that occur

INHIBITION OF CELL DIVISION

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in the cell cortex and that are related to cleavage. And finally, the macromolecular structural orientations of both the cell cortex and the mitotic apparatus can even be dissociated from the submolecular mechanisms responsible for their existence as organized structures (Gross and Spindel, 1960). Both the cleavage furrow and the mitotic apparatus can be made rigid at any stage of operation by a direct influence on the individual hydrogen bonds that cross-link these macromolecular gel structures. It should be re-emphasized that regardless of the degree of independence exhibited by any event discussed earlier in this section - whether it be subsidiary, precursor, or molecular - its impact upon the over-all process of cell division is restricted to the major category within which it is listed in Fig. 1. For example, a continuous and independent cycle of D N A synthesis or of energy yielding respiration cannot move a cell into interphase if a block of any event of mitosis occurs. By the same token, the success of DNA synthesis or of respiration by itself cannot move a cell out of interphase and into mitosis. Thus the progress of cell division from one stage to the next depends upon the concerted effort and success of all of the subsidiary events within each category, and of the previous stage. If this progress is impeded, the synthetic events that have occurred prior to the point of inhibition are not necessarily wasted. Inhibition of chromosomes splitting results in polyteney (Alfert, 1954; Kaufmann et al., 1960) because chromosome reproduction involving duplication in the number of chromonemata has already occurred. Inhibition of chromosome movement by persistence of the nuclear membrane results in polyploidy (Geitler, 1953), because chromosome reproduction and splitting into two chromatids have already occurred. Inhibition of chromosome movement or partitioning by a defective influence on the mitotic apparatus or on cytoplasmic cleavage can also result in polyploidy (Eigsti and Dustin, 1955), or it may result in a binuclear cell depending upon whether the two sets of chromosomes reconstitute into one or two nuclei (Kawamura and Dan, 1958). In addition, the cell cortex does not lose its specific ability to cleave even though cytokinesis is held up for a generation time either at metaphase (Mazia, 1958; Mazia and Zimmerman, 1958) or at furrowing (Marsland, 1958) in echinoderm eggs, for when the blocks are removed, and at the time of the following division, the eggs divide directly into four cells. Thus the cell division process contains a system of inherent and experimentally susceptible check points from which the cell as a whole can be returned to the starting gate. However, the progress of certain biochemical and physiological events, once made, becomes irreversible, and the products of these events may be retained by the aborted cell. The idea of inherent check points or organized units of events in cell division brings up for consideration the correlated concept of “points of no return,” introduced by Mazia (1961b). The cell here is viewed as passing through a series of critical transition steps, each one being prefaced by a decision to make

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SEYMOUR GELFANT

the step. Once the decision has been made the cell is obligated to make the transition, and the step or event can no longer be blocked experimentally. For example, the synthesis of DNA is considered to be a transition event which usually leads the cell into mitosis. Although DNA synthesis can be prevented, once the decision to begin synthesis has been made, it can no longer be prevented experimentally. The other examples of transition events listed include the decision to enter mitosis (in relation to energy production), and the decisions involved in the progress of the cell through the individual stages of mitosis. Thus, the “point of no return” concept proposed by Mazia stresses the inevitable progression of events, whereas the approach in the present review focuses on the inherent and experimentally sensitive check points or categories of check points as shown in Fig. 1, and as analyzed in the preceding discussion. In addition, the individual transition steps listed by Mazia would be viewed as subsidiary events within major categories in Fig. 1 and therefore incapable in themselves of moving a cell from one category to the next. The last analytical approach shown in Fig. 1 deals with the time sequence of cell division as seen from the point of view of DNA synthesis. This measurable sequence or cycle of cell division was originally devised by Howard and Pelc (1953) and by Lajtha et al. (1954) from autoradiographic studies on the timing of DNA synthesis in bean root and in human bone marrow cells. The essential features of this cell cycle have since been confirmed in a wide variety of plant and animal cells both iiz vitvo and iiz vivo (see Stanners and Till, 1960; and references in Gelfant, 1962). The cycle is divided into three interphase periods plus the period of mitosis. It consists of a long postmitotic gap (GI) lasting 10-20 hours, followed by a period of DNA synthesis (S) of from 6 to 8 hours, followed in turn by a short premitotic interval (G2)of about 1-4 hours and finally by a rather rapid period of mitosis ( M ) which lasts about 1 hour. In certain cell populations (Gelfant, 1962) the actuaI period of time spent in G,, or in Gz,may be much longer than the 10-20 hours or 1-4 hours shown in Fig. 1, and this has been indicated by the insertion (Days) or ( D ) in the appropriate positions in the diagram. However, the relative proportions of time spent in G, (more than half the total generation time) or in G, (less than one-fifth) appear to be similar in all cell types (Stanners and Till, 1960; Gelfant, 1962). As indicated above, this scheme for segmenting the process of cell division in time focuses on only one biochemical event, namely, the synthesis of DNA. In a descriptive effort to delimit the period during which this event occurs in interphase, the terms Gap, (G,) and Gap, (G,) have been adopted. This is rather unfortunate because of the possible implication that nothing else of consequence is happening during these “gap periods.” The situation is reminiscent of the use of the term “resting stage” to describe the interphase of cell division when compared to the visibly active stages of mitosis. Nevertheless the real impact of establishing the time sequence of DNA synthesis has been indeed to

INHIBITION OF CELL DIVISION

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counteract the erroneous notion of a “resting stage” and to demonstrate the irrefutable importance of interphase as a physiological stage of cell division. The fact that D N A synthesis can be delimited in time between two non-DNA synthetic periods in interphase presents an additional analytical insight into the question of dissociability of the events of cell division. It also opens a new approach to the study of interphase preparations for mitosis which may eventually lead to a detailed “time map” of the individual biochemical and physiological events as they occur in relation to time in interphase (one such map covering the whole process of cell division has already been drawn by Mazia, 1961b). Meanwhile it has been possible to distinguish inhibition of mitosis that occurred as a result of a block during the GI period {rat liver and bone marrow, and human bone marrow cells (Bollum et nl., 1960; Elson et a/., 1958; Lajtha et al., 1958) 1, or as a result of a block during the S period [mouse intestinal epithelium, hair follicles, epidermis, fibroblasts and HeLa cells (Sherman and Quastler, 1960; Cattaneo et al., 1960; Evensen, 1961; Smith, 1961)}, or as a result of a block during the G2 period of interphase [mouse L-strain and HeLa cells, mouse epidermis, chick fibroblasts, and onion root tip cells (Whitmore et al., 1961; Painter and Robertson, 1959; Devick, 1961; ChPvremont et al., 1960; Das and Alfert, 1961)]. An example of a naturally occurring dissociation of the G, period from the rest of the cell cycle has recently been reported in mouse ear epidermal cells (Gelfant, 1962). A unique population of cells was discovered in which the normal cell cycle is detained after the period of D N A synthesis. These cells do not automatically enter mitosis, but rather remain in the G 2 phase for long periods of time (in readiness for mitosis), and thereby serve as a fast acting renewing system for tissue repair. Thus the procedure of discovering and studying the interphase events in relation to time, and particularly a critical illumination of the events occurring during the G?period should prove to be extremely fruitful. It becomes evident from the preceding analysis of Fig. 1 that the question of itzhibitioiz requires an understanding of the nzerhanisms of cell division, and that inhibition and progress are but two sides of the same problem. Although the major categories and time sequence of events in Fig. 1 are accurate and well established, the subsidiary and precursor events listed are not considered to be complete. Examples of events on this level have been undoubtedly overlooked and others are yet to be discovered. In addition, some of the sub-events may even be slightly inaccurate with regard to their generalized significance in all plant and animal cell types, and also with regard to where they have been placed within a particular morphological category. Nevertheless, the general principles of continuity, dissociability, dependence and independence of events are valid, and they can be used experimentally to analyze the mechanisms involved in either the progress or in the inhibition of cell division. It should be explained that no mention was made of the specific experimental

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SEYMOUR GELFANT

inhibitors that were used to illustrate the general principles discussed above. This omission was deliberate because the purpose of the preceding analysis was to establish a potential pattern of investigation for studying various problems of cell division. The questions of mechanism of action and of universal effects of inhibitors will be taken up in the following sections of this review by discussion of the literature and of experiments performed. 111. Experimental Systems Used

The following experimental systems were used in the present report to evaluate the effects of various cell division inhibitors.

A . MOUSEEAR EPIDERMISiiz Vitro This system, devised by Bullough and Johnson (195 I ) , and used extensively by Bullough and by the present author (see references, Gelfant, 1960c), offers a number of advantages for studying cell division. Cutting of the ear prior to its incubation it? vitro specifically induces epidermal cells that were in the G, period (Gelfant, 1962) to enter mitosis in vitro (Gelfant, 1959a). In addition to the cutting stimulus, the expression of epidermal mitosis it? vitro also depends upon the composition of the culture medium (Gelfant, 1 9 6 0 ~ )Thus, . once mitosis has been initiated in G, by cutting, one can study the various culture conditions that might be involved secondarily in the movement of a cell from the G, period of interphase into mitosis iiz vitro. And if the culture conditions are kept constant, this system provides a population of dividing epidermal cells in vitro to test the effects of inhibitors on any stage of mitosis. The essential features of this in vitvo procedure involve the use of adult male mice. Their ears are cut into small pieces and are incubated in Warburg flasks in a Krebs-Ringer phosphate-buffered saline medium at 3 8 O C. Substrates, inhibitors, or other compounds may be added to the culture medium. The ear fragments are incubated for 1 hour to allow all mitoses originally present in the epidermis to pass beyond metaphase. At this point the experimental period begins. If one is studying culture conditions that might influence the expressiorr of mitosis itz zlitro, colchicine is tipped into the main vessels of the Warburg flasks and incubation is continued further for 4 hours. The effects of the various substrates, compounds, or culture conditions in the different flasks on the development of epidermal mitosis are determined in histological sections by counting the number of metaphase figures arrested by colchicine during the 4-hour experimental period. On the other hand, if one is testing the ability of inhibitors to inhibit mitosis in metaphase for example, the culture conditions are kept constant in the different flasks and the inhibitors are applied in the same manner as colchicine. They are tipped into the main vessels of the Warburg flasks, allowed to act for 4 hours, and their effects are then compared with colchicine, again by counting the number of epidermal metaphase figures arrested during the experimental period in

INHIBITION OF CELL DIVISION

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vitro. Thus in the first instance, colchicine is used as a tool to demonstrate an interphase effect, whereas in the second case colchicine is used as a comparative metaphase inhibitor. There are a number of factors, recently reviewed by Bullough and Laurence (1961), that may lead to variable or to abnormal results if this particular itz vitro method is used improperly. The main sources of error involve the use of female animals, animals that have been stressed, or whose ears have been irritated or wounded. All of these conditions lead to an abnormally high rate of epidermal mitotic activity in vitro (Bullough and Laurence, 1961). It should be stated that the present author has always used adult male unstressed mice with normal healthy ears (for a detailed description of our technique see Gelfant, 1959a). The evidence for this point can be found in the relatively low (normal) mitotic counts recorded by the author whenever this it2 vitvo method was used (Gelfant, 1958a,b; 1959a,b; 1960a,b,c,d). For an over-all discussion of culturing different types of epidermal cells i t 2 vitro see the excellent review by Matoltsy (1960).

B.

MOUSEBODYSKIN EPIDERMIS in Vivo

The if, vitro effects of various cell division inhibitors were further evaluated iii uivo using stimulated mouse body skin epidermis. Epidermal mitosis was stimulated in a dorsal area of skin by plucking the hairs from resting follicles, or by making a simple wound in the skin with a scalpel blade. Both procedures result in a high degree of mitotic activity 48 hours after epidermal damage (Bullough and Laurence, 1960a,b). And if the situation in mouse body skin epidermis is similar to mouse ear epidermis iir vir~o,we can also assume that plucking or cutting the skin induces epidermal cells to enter mitosis in vivo from the G , period of the cell cycle (Gelfant, 1962). Stimulated mouse skin epidermis thus provides an itz vivo population of dividing epidermal cells to test the effects of cell division inhibitors. For discussions of epidermal mitotic activity in relation to hair growth and to wounding, and for detailed descriptions of techniques see reports by Chase (1954), Chase et d.(1953), Argyris (1954, 1956), and Bullough and Laurence (1960a,b). And for general reviews on the properties of epidermal cells in viuo; on the physiology and on wound healing of skin see reviews by Matoltsy (1960), Montagna (1956, 1961), and Johnson and McMinn (1960). C. RAT UTERUS in Vivo The in uiuo effects of a number of inhibitors were analyzed further in an entirely different system, the rat uterus. Some of our previous data on the inhibition of uterine growth and cell division (Gelfant et a/., 1955) were used for this purpose. In this system a controlled comparison can be made of the effects of an inhibitor on a variety of uterine tissues: epithelium, gland, stroma, and muscle. In addition mitotic activity can be correlated with changes in total uterine

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SEYMOUR GELFANT

growth (dry weight). Uterine growth and cell division are stimulated experimentally in ovariectomited rats by the administration of estrogen (for details of techniques see Gelfant et al., 1955). The stimulated uterus thus provides a number of populations of actively dividing cells. The effects of interphase inhibitors are determined by administering colchicine and counting the number of cells that enter mitosis over a 6-hour period. Colchicine is used in the same manner to correlate mitotic activity with either the stimulation or with the inhibition of total uterine growth.

IV. Synopsis of Inhibitors Used The following nine inhibitors were studied experimentally. A.

COLCHICINE

Colchicine, which is an alkaloid that is isolated from the plant Colrhicum autumnale, is a universal mitotic inhibitor, effective in plant and animal cells both it, zhro and in i)ivo (for the most comprehensive treatise on this subject see Eigsti and Dustin, 1955; other illuminating articles by LettrC, 1952, 1954; Benitez et al., 1954; Levine, 1951; Padawer, 1960). Colchicine inhibits mitosis in metaphase by disorienting the structural organization of the asters and the spindle (Inoui, 1952; Gaulden and Carlson, 1951; Sauaia and Mazia, 1961). The chromosomes are thus unable to move and may be arrested in metaphase for as long as 10 hours, after which the cell either degenerates or recovers from the effects of colchicine (Eigsti and Dustin, 1955). Although a number of theories have been proposed to explain the action of colchicine on the spindle (Lettri, 1952, 1954; Padawer, 1960; Eigsti and Dustin, 1955), its exact mechanism of action remains unknown (Benitez el al., 1954; Biesele, 1958; Sauaia and Mazia, 1961). The particular colchicine derivative used in the present study was N-desacetylN-methylcolchicine ( Colcemid2 or demecolcine) . Colcemid is reportedly less toxic (Schar et al., 1954) and somewhat more effective than colchicine (Meier et al., 1954). Also, Bullough and Laurence (1961) indicate that different colchicine preparations may vary in potency from sample to sample, and they therefore recommend the use of Colcemid to arrest mitosis in metaphase in studies on mouse ear epidermis in uitro. It should be pointed out that the present author has always employed colcemid in studies on mouse ear epidermis, but has preferred calling it by the more well known generic name, colchicine. Perhaps the greatest value of colcemid over colchicine is its preferential action on the mitotic apparatus of sea urchin eggs (Sauaia and Mazia, 1961). Colcemid first attacks the asters, which allows for the possibility of isolating spindles without 2

Ciba Laboratories, Ltd.

INHIBITION OF CELL DIVISION

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asters, and provides material to study the biochemistry and physiology of the mitotic spindle. B. PODOPHYLLIN~ Podophyllin, which is a lignan that is isolated from the plant Podophpllunz peltaturn, resembles colchicine in its effects on mitosis. It inhibits mitosis in metaphase in a wide variety of plant and animal cell types (Biesele, 1958), as does colchicine, by destroying the structural organization of the mitotic apparatus (Cornman and Cornman, 1951) and preventing chromosome movement. Although a great deal is known about the general pharmacological effects and chemical composition of podophyllin (Kelly and Hartwell, 1954) and about its molecular structure-activity pattern (Padawer, 1961), almost nothing is known of its mechanism of action either on the spindle in dividing cells (Biesele, 1958), or on the morphology of nondividing cells (Padawer, 1961). C.

VINCALEUKOBLASTINE (VLB)4

Vincaleukoblastine, the alkaloid which has been recently isolated from the plant Vima r.oJea Linn (Noble et al., 1958), is currently receiving maximum attention because of its antitumor actions in laboratory and clinical studies (Johnson et a/., 1960, 1961; Cutts et al., 1960; Warwick et al., 1960; Hodes et al., 1960; Hertz et al., 1960; also see section on Chemotherapy which deals with Canadian Cancer Conference, 1961). Our interest in this compound arose from the preliminary report by Palmer and associates (1960) indicating that VLB arrested mitosis in metaphase in cells grown in vitro. It has since been shown that VLB also inhibits mitosis in metaphase in t h o , and that it appears to be a more efficient metaphase inhibitor than colchicine (Cutts, 1961; Cardinali et a/., 1961). Almost all of the studies on VLB are concerned with its antitumor properties; its action on the spindle during metaphase is inferential (Palmer et a/., 1960; Cutts, 1961), and nothing has been reported on the biochemical mechanism of action by which it produces metaphase arrest. Indeed, it has been suggested that the oncolytic biochemical action of VLB (interference with glumatic acid metabolism) may even be unrelated to the phenomenon of metaphase arrest (Johnson et nl., 1961).

D. CHLORAL HYDRATE; Chloral hydrate, a compound which is a narcotic, also has a colchicine-like effect on the spindle and inhibits mitosis in metaphase (Ostergren, 1944; Sentein, 1947). Although chloral hydrate is classified as a generalized metaphase spindle 5 Podophyllin (U.S.P.) obtained from Nutritional Biochemicals Corp. was used in the present study. 4 The compound used in the present study, vincaleukoblastine sulfate, was generously supplied by Eli Lilly and Company. +. The compound used in the present study was obtained from Eastman Organic Chemicals.

14

SEYMOUR GELFANT

inhibitor (Ludford, 1953; Biesele, 1958), its action has been studied only in a limited number of cell types (primarily in segmenting urodele eggs and onion root tip cells). Sentein (1960, 1961) is currently making a detailed analysis of the cytological effects of chloral hydrate and various derivatives, but has not yet determined its biochemical mechanism of action. Perhaps the most valuable and dramatic effect of chloral hydrate has been its selective action on the spindle in isolated grasshopper spermatocytes (Ris, 1949). Chloral hydrate specifically inhibits spindle elongation but not poleward movement of chromosomes in grasshopper spermatocytes, and Ris has thus been able to elucidate the two movements involved in the separation of chromosomes during anaphase.

E. MERCAPTOETHANOL~ Mercaptoethanol is a sulfhydryl containing compound (monothioethylene glycol) which has recently achieved prominence as a metaphase inhibitor through its use as an experimental tool in the studies of Mazia and co-workers on echinoderm eggs (Mazia, 1958; Mazia and Zimmerman, 1958; Mazia et al., 1960; Bucher and Mazia, 1960). Mercaptoethanol arrests mitosis in metaphase (in a colchicine-like manner) by disrupting the fibrous organization of the spindle. This disorganization is, however, only macromolecular, because the structural orientation of the entire mitotic apparatus is readily restored within minutes after removal of mercaptoethanol (Mazia and Zimmerman, 1958; Mazia, 1961b, p. 237). It is therefore postulated that mercaptoethanol acts by loosening the major macromolecular bonding of the spindle to a point where it no longer holds together, but not to a point where the underlying molecular gel structure of the mitotic apparatus is destroyed. Although a number of suggestions have been made regarding the biochemical mechanism of action of mercaptoethanol on the spindle (interference with sulfur bonding, or indirectly with energy requirements) its exact mechanism of action remains unknown. The same is true of its inhibitory actions on regenerating Acetabularia (Brachet, 1958), and on morphogenesis, nuclear division, and cleavage in synchronized Tetwhymeua (Holz, 1958). F. MALEURIC ACID7 Maleuric acid is a compound (N-carbamylmaleamic acid) of which the mitotic inhibitory actions have been studied in onion root tip and Ehrlich ascites tumor cells (Okada and Roberts, 1958, 1960; Sisken et al., 1959). According to these authors, maleuric acid has at least three effects on cell division: it inhibits DNA synthesis - as seen by autoradiographic tritium labeled thyrnidine incorporation; it inhibits the progression of cells from the G, period of interphase into 0 The compound used in the present study, 2-mercaptoethanol, was obtained from Eastman Organic Chemicals. 7 The compound used in the present study was supplied by Naugatuck Chemical Co.

INHIBITION OF CELL DIVISION

15

mitosis - as seen by the fact that labeled interphase nuclei do not enter mitosis when maleuric acid is applied after labeling; and it inhibits mitosis in metaphaseas seen by the piling up of dividing cells in the metaphase stage. Although the mechanism of action of this multiple inhibitor has been studied in relation to protein synthesis, RNA and DNA synthesis, and sulfhydryl metabolism, the question of the mechanism of action of maleuric acid remains unsolved (Okada and Roberts, 1960). G. NITROGEN MUSTARD' The nitrogen mustards {RN(CH,CH,CI) are a class of compounds derived from mustard gas [S(CH,CH,Cl),]. Studies of the many biological and biochemical effects of the nitrogen mustards seem to place them in the following categories: (a) Mutagenic agents - those producing gene mutations {originally observed in Drosophda (see Auerbach, 1950, 1951; Boyland, 1954)j. (b) Cytotoxic agents - those producing chromosomal aberrations {first observed in Tradescaiztia pollen grains (see Koller, 1958) 1. (c) Cairiiiogenic aizd carcinostatic ageizts - those capable of both induction and temporary remission of experimental tumors (for carcinogenic action see Boyland, 1952; for anticancer actions see reviews by Skipper, 1953, Skipper and Thomson, 1955; Skipper and Bennett, 1958; Bergel, 1955; Mandel, 1959; Montgomery, 1959; Lane and Kelly, 1960; Rutman et al., 1961a,b). ( d ) Alkylatiizg agents - activated electrophilic compounds capable of combining with a variety of important biochemical nucleophilic groups such as amino and mercapto groups, and the anions of organic and inorganic acids. In the case of the mustards, one of the chloroethyl groups loses its chlorine and is converted into an ethylene imine and thus becomes activated. O n reaction, the three membered ring of the ethylene imine is opened and the ethylene group attaches to the reacting compound (for excellent reviews and discussions on the alkylating actions of nitrogen mustards see FriedenwaId, 1951; Bergel, 1955; Alexander and Cousens, 1958; Ross, 1958; Montgomery, 1959; Walker and Watson, 1961; Rutman et a/., 1961a,b; Trams et al., 1961a,b). (e) Nucleic acid-alkylatiag agent interactions. Studies on this subject fall into a separate category because of their efforts to establish a common explanation for the many biological effects of the nitrogen mustards. For reports dealing with the inhibition of DNA synthesis see Bodenstein and Kondritter (1948), Herriot (1951), Goldthwait (1952), Ultmann et a/. (1953), Drysdale et al. (1958), and Konigsberg et al. (1960) ; for the inactivation of the transforming principle of DNA see Zamenhof (1956). For studies of the specific alkylating reactions of nitrogen mustard on nucleic acids both iu zitro and jiz Z ~ J O :for 8 The compound used in the present study was methyl bis (P-chloroethyl) arnine hydrochloride (HNZ), obtained as Mustargen from Merck and Co.

16

SEYMOUR GELFANT

degradation of D N A see Butler et al. (1950, 1951); for esterification of the phosphate groups of D N A and RNA see Stacey et al. (1958); for reactions with the purine and pyrimidine bases of D N A see Wheeler et al. (1955), Brookes and Lawley (l960), Walker and Watson (1961), and Rutman et al. (1961a,b); for an over-all discussion of alkylating reactions see Kit (1960) ; and for a most valuable and controversial point of view on the interaction of alkylating agents with nucleic acids see papers by Trams et al. (1961a,b) and Rutman et al. (1961~). ( f ) Antimitotic agents -chemical and physical agents that inhibit cell division. For some of the original observations on the antimitotic actions of nitrogen mustard see Bodenstein (1947) and Bodenstein and Kondritzer (1948) (in amphibian embryos) ; Friedenwald et al. (1948) and Friedenwald (195 1) (in corneal epithelium) ; Loveless and Revel1 (1949) and Novick and Sparrow (1949) (in plant root tips); Hutchens and Podolsky (1948) (in sea urchin eggs) ; Hughes (1950) and Rinaldini (1952) (in tissue cultures) ; Daniels (1952) and Kimball et al. (1952) (in protozoa). In spite of all of the studies alluded to above, there is no clear-cut agreement as to how nitrogen mustard inhibits ceIl division. To begin with, there are a few conflicting reports concerning the point of inhibition, Although most studies indicate that nitrogen mustard prevents the onset of mitosis and is an interphase inhibitor (Bodenstein, 1947; Freidenwald et al., 1948; Novick and Sparrow, 1949; Rinaldini, 1952; Gelfant et ul., 1955), there are a few reports in which nitrogen mustard inhibits mitosis in metaphase [(Hughes, 1950; Cardinali, 1949) (in chick tissue cultures)]. Even if we accept, by weight of the evidence, that nitrogen mustard is an interphase inhibitor, the question of its mechanism of action is still an issue. There is a strong body of evidence showing that nitrogen mustard inhibits D N A synthesis: Bodenstein and Kondritzer (1948) upon chemical determinations of D N A and RNA synthesis in amphibian embryos; Herriott (1951 ) - after D N A and RNA determinations in E. coli; Goldthwait (1952) - after measurement of the incorporation of labeled precursors into the bases of D N A and RNA of the small intestine of the rat; Lowrance and Carter (1 950) - after measurement of P3? incorporation into DNA and RNA of bone marrow, spleen, and thymus; Swift (1953) - after cytophotometric determination of D N A contents in individual nuclei of onion root meristem; and more recently, Konigsberg et al. (1960) - after both chemical extraction and incorporation of precursors into D N A of chick muscle cells it2 t h o . On the other hand, there is an equally strong and growing body of evidence to show that nitrogen mustard does not inhibit D N A synthesis; indeed, there are some cases in which it appears to stimtrlate D N A synthesis. The first important indication of this view came from the report of Ultmann et al. (1953). These authors showed that although nitrogen mustard inhibits cell division, it

INHIBITION OF CELL DIVISION

17

does not interfere with the usual increase in nucleic acid concentration in regenerating rat liver. As a matter of fact, the level of DNA and RNA in the nitrogen mustard treated liver cells whose division was blocked was even higher than in the controls. It was therefore concluded that mustard inhibition of mitosis was not mediated primarily through an inhibition of nucleic acid synthesis. A similar conclusion has recently come out of the biochemical studies of Trams et ul. (1961a,b) on regenerating liver and ascites tumor cells. These workers found that the alkylating attachments of mustards to polymerized DNA do not account for the cytotoxic effects of such compounds, and second, that the actions of nitrogen mustard and other alkylating agents on the biosynthesis of nucleic acids resulted in stimulation as well as inhibition of precursor utilization, depending upon which precursor was being studied. In another recent report by Rutman et al. ( 1 9 6 1 ~ )on Ehrlich ascites tumor cells, it was also found that alkylation of DNA by nitrogen mustard bears no relationship to cytotoxicity. These authors come close to concluding that the sensitive sites of nitrogen mustard inhibition of cell division are to be found elsewhere than in the DNA per se. There are, finally, a number of other observations that question the mitotic inhibitory action of nitrogen mustard as being due to inhibition of DNA synthesis. Davidson and Freeman (1955) observed an apparent stimulation of P3? incorporation into DNA in nitrogen mustard treated mouse tumor cells; Brachet (1957, p. 203) also reports a case of stimulation of DNA synthesis in the male hemikaryon nucleus of the frog egg fertilized with nitrogen mustard treated sperm; and Podolsky and Hutchens (1954) noted that synthesis of nucleic acids (in the plantlike flagellate Chiionzoim) is not inhibited by nitrogen mustard during the period in which cell division is most effectively inhibited. Thus there are two conflicting sets of observations concerning the action of nitrogen mustard on DNA synthesis as the mechanism of inhibiting cell division. It is, however, possible to resolve these two points of view if we assume: (1) that nitrogen mustard inhibits cell division during the G 2 period of interphase, after DNA synthesis has occurred; ( 2 ) that it does not prevent the synthesis of DNA in those cells that have not as yet reached the G, period; and ( 3 ) that further DNA synthesis ceases once DNA doubling has taken placed in the nitrogen mustard blocked cells. These assumptions would then explain the apparent inhibition of DNA synthesis and of the accumulation of extractable DNA, if the comparison between the nitrogen mustard treated cells and the continuously synthesizing control cells was made after doubling had already taken place in the mustard treated system. By the same token, these assumptions would also explain the lack of inhibition of DNA synthesis, or even the apparent stimulation of DNA synthesis, if the comparisons were made at various points before all of the nitrogen mustard treated cells had synthesized their DNA. A comparison of the DNA content per cell in nitrogen mustard blocked cells that have dcubled their DNA - with a control system in which the cells are constantly

18

SEYMOUR GELFANT

leaving the 4C (double) DNA condition to enter mitosis -would give the appearance of a greater accumulation of DNA in the treated cells than in the controls. This analysis of the mechanism and of the point of inhibition of nitrogen mustard will be further alluded to in a later section (Section VII,B) of this report.

H. AMINOPTERIN Aminopterin (4-aminopteroylglutamic acid) is a substance that interferes with the conversion of folic acid (pteroylglutamic acid) to folinic acid (tetrahydrofolic acid). Folinic acid is intimately involved in the synthesis of purines and of thymine necessary for the formation of nucleotides and nucleic acids. Hence, folic acid antagonists such as aminopterin or amethopterin (4-aminoN1°-methylpteroylglutamic acid) inhibit nucleic acid synthesis and have profound biological effects. Most of the studies on this subject have been concerned with the effects of folic acid analogs on the conversion of folic acid to its various functional folinic, coenzyme forms and with the catalytic roles of these coenzyme derivatives in purine and thymine biosynthesis. There has also been a considerable research effort to determine the usefulness of folic acid antagonists as anticancer agents. For some informative and valuable discussions, reviews, and recent references to these areas see Petering (1952), Huennekens et at. (1958), Nichol et al. (1955), Hakala et al. (1961), Skipper et al. (1950), Skipper and Bennett (1958), Darrow et al. (1960), Dinning and Young (1960), Eidinoff et d. ( 1961), Welch (1961), and especially Handschumacher and Welch (1960). If folic acid antagonists interfere with DNA and RNA synthesis, they should exert a differential effect on dividing cells and on nondividing cells that are or are not synthesizing D N A in preparation for division. This point was cleverly demonstrated by Barton and Laird (1957) who showed that amethopterin inhibited DNA and RNA synthesis only in tissues engaged in active cell division, such as in regenerating rat liver, in the spleen and small intestine, but not in tissues that were engaged in nonmitotic growth, such as intact liver recovering from fasting. Their results further indicate that the action of folic acid antagonists on RNA synthesis is also restricted to the process of cell division. In addition, Barton and Laird observed that amethopterin administered for short periods of time was ineffective but that prolonged periods of administration were required to produce inhibition of nucleic acid synthesis. This point will be alluded to later because it illustrates the too often overlooked reason for administering folic acid antagonists in viuo, namely, to produce a vitamin deficiency comparable to the gradual depletion of folic acid by dietary means. Another study most relevant to these issues is the report by Grant (1960) on the influence of folic acid analogs on the embryonic development of R a m pipiem. To begin with, Grant confirms the observation that the action of antifolic acid drugs is directed chiefly against tissues in which there is active cell division, by showing

INHIBITION OF CELL DIVISION

19

that embryonic regions exhibiting high mitotic activity (neural folds, somites were most sensitive to the analogs, whereas notochord and endoderm exhibited the least sensitivity. Second, there was an excellent correlation between the concentration of embryo folk acid activity at any specific stage, as measured by microbiological assay, and analog sensitivity at the same stage. When the level of total folk acid activity is maximum, the embryo is least sensitive to the analog, and at the peak of analog sensitivity, the amounts of tissue folic acid compounds are at a minimum. This illustrates again that folic acid antagonist action in vivo should be viewed first in terms of producing a vitamin deficiency and in relation to the existing tissue levels of folic acid, and then in terms of folk acid transformations and the biosynthesis of thymine and purines. Finally, Grant noted that although amethopterin inhibited D N A synthesis, it had no effect on the incorporation of precursors into RNA or on the amount of RNA per embryo. This latter observation points out the substantial selective depression by folic acid antagonists of D N A thymine synthesis. This has also been noted by other workers (see Handschumacher and Welch, 1960; and Eidinoff et al., 1961). The fact that antifolic acid agents can interfere with purine synthesis of both nucleic acids, but that they appear to have a preferential effect on D N A thymine synthesis, gives rise to the question of what stage of cell division should be inhibited by these compounds. At this point the literature on the mitotic actions of folic acid antagonists becomes somewhat irrational. The consensus of opinion has been that aminopterin, for example, acts as a nze;aphase inhibitor. This opinion is derived mainly from short-term iiz vitro studies, particularly from the work of Jacobson (1952, 1954a,b; Jacobson and Cathie, 1960a) on chick embryo fibroblasts and osteoblasts grown in tissue culture. Similar observations have also been recorded by Hughes (1950) in chick tissue cultures, Benitez et al. (1954) in rat fibroblasts in culture, and by Biesele (1954) in cultures of embryonic mouse skin. The “reasonable” interpretation of the metaphase arrest caused by aminopterin as stated by Jacobson (1954a) and by Biesele (1958, p. 159) involves the action of antifolics on the ribonucleoprotein material released from the chromosomes during anaphase (Jacobson and Webb, 1952). This appears to be related to the contraction of the chromosomal fibers and the separation of chromatids during anaphase. The opinion as recently expressed by Jacobson and Cathie (1960a), “Thus the arrest of cell division in metaphase by folic acid antagonists can be viewed as a general phenomenon,” continues to ignore a number of in vivo reports showing that aminopterin inhibits cell division in interphase in mouse intestine (Dustin, 1950; Grampa and Dustin, 1952, 1953) and in rat uterus (Gelfant ef al., 1955). Moreover, Grampa and Dustin and Gelfant ef al. employ colchicine to demonstrate that aminopterin treated cells never enter into mitosis to reach metaphase. The question of metaphase inhibition by aminopterin as observed in tissue culture was examined briefly by the present author (Gelfant, 1958b). It was pointed out that a wide variety

20

SEYMOUR GELFANT

of chemical substances, including distilled water, cause metaphase inhibition in dividing chick cells grown in culture, and that it seemed unlikely that aminopterin was acting as a chemically defined inhibitor within the short period of 15 minutes in which it was being studied iiz v i t ~ o .However, except for the lone contradiction by the present author, the view that folic acid antagonists are metaphase inhibitors continues to be propagated and has received widespread publication (see references above; in addition, see reviews by Ris, 1955 ; Swann, 1957; Bass, 1959; Biesele, 1961).

I. ACTIDIONE~ Actidione, a compound that is a cycloheximide antifungicide (antibiotic), has been used exclusively by Wilson and collaborators as a mitotic inhibitor in excised pea roots (Bowen and Wilson, 1954; Hadder and Wilson, 1958; Wilson, 1960). According to these workers, Actidione inhibits mitosis in prophase by preventing the breakdown of the nuclear membrane. The biochemical mechanism of action here is completely unknown. In addition to Actidione, a number of other antibiotics such as streptomycin, streptothricin, Chloromycetin, Aureomycin, and Terramycin also inhibit pea root mitosis in prophase. The question of whether Actidione has antimitotic effects on other experimental systems has not been studied. For some of the more recent studies on the antimitotic and anticancer activity of chemical compounds see: “Mitotic Poisons and the Cancer Problem” by Biesele (1958) (the most comprehensive and valuable book) and other subsequent reviews by Biesele (1960, 1961, 1962); see also Turchini and Sentein (1960). Other relevant references include the reviews and reports by Wood (1959), Bass (1959), Wilson (1960), Lettre (1960), D’Amato (1960), Trowell (1960), Nichol (1960), Dixon et nl. (1961), Levintow and Eagle (1961), Goldin et nl. (1961), and ChPvremont (1961). V. Effects of Various Inhibitors on Mitosis in Vitro A.

METAPHASE INHIBITORS COMPARED

WITH

COLCHICINE

In Table I the metaphase inhibitory effects of each of the compounds discussed in the previous section were compared with colchicine. In addition, one other compound, agmatine sulfate1‘’was tested because of its reported action of accelerating the rate at which cells pass through prometaphase {in grasshopper neuroblasts (St. Amand et al., 1960)]. As explained in Section III,A, mouse ear fragments cultured ill vi1r.o provide a population of dividing epidermal cells that can be routinely arrested in metaphase by colchicine. Thus if any of the compounds tested are metaphase inhibitors, their action on epidermal mitosis compound used in the present study was supplied by the Upjohn Co. Nutritional Biochemicals Corp.

9 The la

21

INHIBITION OF CELL DIVISION

should prove similar to that of colchicine. The effects of the various inhibitors were also studied after a short 1-hour exposure period to determine any transient effects that might elude observation. These results are not included because there were no qualitative differences between the effects of an inhibitor after 1 hour and after the normal 4-hour exposure period i i z zdm. TABLE I METAPHASE INHIBITION OF MITOSIS I N MOUSEEAREPIDERMIS in Vitro" Experiment 1'

Experiment 2

Inhibitor

No. mitoses' (metaphase)

(0.001-0.01 mM)

None Colchicine Mercaptoethanol Chloral hydrate Actidione

0.2 4.1 0.1 0.5 0.1

Inhibitor

No. mitoses (metaphase)

None Colchicine Podophyllin Am inopterin Nitrogen mustard

0.3 3.8

(0.001-0.01m M )

Experiment 3 Inhibitor (0.01-5.0 mM) None Colchicine Maleuric acid Agmatine

2.8

0.2 0.2

Experiment 4 No. mitoses (metaphase) 0.2 3.9

Inhibitor

(0.001-0.05 mM)

None Colchicine Podophyllin Vincaleukoblastine

0.1

0.9

No. mitoses (metaphase) 0.0

4.9

5.7

6.5

Experiment 5 Inhibitor

(0.01 m M )

None Actidione Agmatine Mercaptoethanol (ME) Vincaleukoblastine (VLB) Nitrogen mustard ( H N 2 ) a

No. mitoses (metaphase) 0.2

0.0

0.4 0.6 4.6 0.1

Colchicine (0.01 mM) (0.01 m M )

+ inhibitor

Colchicine alone Colchicine+actidione Colchicine+agmatine Colchicine+ME ColchicineSVLB Colchicine+HNZ

No. mitoses (metaphase) 4.8 0.0 6.7 4.4 2.3 3.5

Effects of various inhibitors compared with colchicine.

' Five adult male mice were used for each experiment.

Each figure represents the average number of mitoses (arrested in metaphase) per cm. unit length of epidermis in 5 ear fragments incubated for 4 hours at 38'C. in a saline medium with 0.002 M glucose.

The first 4 experiments in Table I show that only colchicine, podophyllin, and vincaleukoblastine are capable of arresting mitosis in metaphase in mouse ear epidermis cultured in ztitro. It would appear, then, that these three compounds are general metaphase inhibitors in z~jtr'o and that mercaptoethanol, chloral hydrate, actidione, aminopterin, nitrogen mustard, maleuric acid, and agmatine are not. The results in experiment 5 indicate that epidermal cells are indeed

22

SEYMOUR GELFANT

passing through metaphase. In most of these cases the inhibitor has failed to arrest mitosis in metaphase, because when colchicine is added in the presence of the ineffective inhibitor (see agmatine, mercaptoethanol, and nitrogen mustard) metaphase arrest occurs. However, this is not the case with actidione, for this compound apparently prevents epidermal cells from entering mitosis iiz vitro. In addition, there is some indication that agmatine may accelerate the speed at which cells reach metaphase, because the number of mitoses arrested by colchicine in the presence of agmatine is greater than with colchicine alone. B.

ENERGYMETABOLISM INHIBITORS

The next question, considered in Table 11, involves the effects of energy metabolism inhibitors on the movement of a cell from interphase into mitosis. This can be studied adequately only in in vitro systems such as in developing sea urchin egg cells (Krahl, 1950; Swann, 1953, 1957), synchronized Tetrabymetla cells (Zeuthen, 1958), or mammalian cells cultured iii zlitro (Bullough, 1952; Gelfant, 1960c; Levintow and Eagle, 1961). In the present study on mouse ear epidermis, cells that enter mitosis in vitru are arrested in metaphase by colchicine. The number of arrested mitoses is then used to demonstrate the impact of an inhibitor during interphase. By studying only the results of experiment 1 in Table 11, it would appear that inhibitors of glycolysis (iodoacetate, fluoride), the citric acid cycle (malonate), and the cytochrome system (azide) depress mitotic activity because they interfere with the conversion of glucose (in the medium) to energy necessary for mitosis. Such conclusions have indeed been made (Bullough, 1952, 1955), but they have also been challenged by the present author (Gelfant, 1 9 6 0 ~ )as is illustrated by experiments 2, 3, and 4. These experiments show that the classical inhibitors of carbohydrate metabolism are not acting in a specific biochemically defined manner A ,.elution to epidermul mitosis in vitro because: (a) the mitotic block imposed by glycolytic inhibitors cannot be bypassed by pyruvate or lactate; (b) a wide variety of metabolites, antimetabolites, and compounds generally unrelated to carbohydrate metabolism (adenine, 8azaguanine, phenylalanine, p-chloromercuribenzoate, and diisopropyl fluorophosphate) also inhibit epidermal mitosis in interphase; and (c) the effects produced by an inhibitor, whether on mitosis alone or on epidermal damage are irreversible. On the basis of these results we concluded (Gelfant, 1960b,c) that carbohydrate metabolism inhibitors cannot be used to prove a relationship between energy production and epidermal mitosis in vitro because their effects (in general) are nonspecific, toxic, and irreversible. One aspect of our technique illustrated in experiment 4, has recently been criticized by Bullough and Laurence (1961). This concerns the detrimental effects of a nitrogen gas phase (anaerobiosis) on epidermis. These authors claim that the toxic effects observed in our experiments are the result of using impure

23

INHIBITION OF CELL DIVISION

nitrogen. They do not, however, present any experimental evidence to substantiate this claim. It should be pointed out first of all, that our conclusions concerning carbohydrate inhibitors and mitosis are based not only upon nitrogenanaerobiosis, but also upon a wide variety of experiments - as shown in Table 11. Second, even a gas phase of carbon monoxide is toxic to mouse ear epidermis SPECIFICITY

OF

TABLE I1 ACTIONOF ENERGY METABOLISM INHIBITORS IN MOUSEEAR EPIDERMIS in vitvo"

Experiment 1 :

ON

MITOTIC ACTIVITY

Experiment 2: Specificity A. Glycolytic inhibitors

Carbohydrate inhibitors

No. mitoses Inhibitor None Iodoacetate (IOA) Fluoride Malonate Azide

No. mitoses' 4 .O 0.1 0.3

0.3

0.0

None Adenine 8-Azaguanine Phenylalanine P-CMB DFP

Alone

None Glucose Fructose Pyruvate Lactate

I .o 5 .O

Inhibitor IOA or NaF 0.3 0.2 0.2

4.0 3.0 3.0

0.9 0.2

Experiment 4: Specificity C. Recuperative capacity

Experiment 3: Specificity B. Noncarbohydrate inhibitors Inhibitor

Substrate

Inhibitor, ( 5 hrs. )

No. mitoses

wash -3

None Malonate Azide Adenine 100% Nitrogen Carbon monoxide (dark)

5 .O 0.1

0.1

0.3 0.1 1.5

No inhibitor, (4 hrs.) 2 .o 0.G

0 .o 0.1

Necrotic Necrotic

' Data from various experiments were combined and modified after Gelfant ( 19GOb,c).

* Each figure represents the average number of mitoses (arrested in metaphase by colchicine) per cm. unit length of epidermis in 5 ear fragments incubated for 4 hours at 38" C. in a saline medium containing glucose.

in v h o (experiment 4). This does not mean that carbon monoxide (a specific cytochrome inhibitor) or a nitrogen gas phase (anaerobiosis) does not curtail energy production. These gases are nevertheless toxic in this experimental system, and therefore any allusions here to their specific biochemical effects in relation to energy production and mitosis must be disregarded. C.

HORMONES

The situation concerning the inhibitory effects of hormones on mitosis in mouse ear epidermis in d r o is quite similar to the one just discussed. In this

24

SEYMOUR GELFANT

case Bullough (1955) establishes a theory of hormonal control of cell division which is applicable to hormones that influence carbohydrate metabolism. More specifically, the glucokinase enzyme system i s considered to be the one pivotal and rate-limiting reaction by which most hormones exert their influence on mitosis. Thus, growth hormone, for example, is a mitotic inhibitor because it TABLE I11 OF ACTIONOF HORMONES (INHIBITORS) ON MITOTIC ACTIVITY SPECIFICITY IN MOUSEEAREPIDERMIS v/it?'O" ~

Experiment 1:

Experiment 2 : Specificity A. Metal Contaminants

Hormones (inhibitors) ~~

~

Trace quantity metal ( 1 0 - 7 M ) None FeCI, CrCI, AgCl AICI.,

No. mitoses'

Hormone None Insulin Growth hormone Adrenaline DOCA

7.1 2.9 1.6 0.1 1.9 ~~

~~

No. mitoses 6.1 2.5 2.3 2.8 2.7

~~~~

Experiment 3 : Specificity B. Glucokinase reaction Substrate

Alone ~

None D-Glucose D-Fructose D-Galactose o-Mannose r-Arabinose D-Ribose D-Xylose

~

0.2

3.5 3.5 3.5 3.5 1.5 1.5

1.5

~

With insulin

With growth hormone

0.2 1.o 1.O

0.2 1.o 1 .o 1.o

~

1.o

1.o 0.2 0.2 0.2

1.o 0.2

0.2 0.2

Data from various experiments were combined and modified after Gelfant ( 1960d). figure represents the average number of mitoses (arrested in metaphase by colchicine) per cm. unit length of epidermis in 5 ear fragments incubated for 4 hours at 38" C . in a saline medium containing glucose. a

' Each

inhibits glucokinase and prevents the conversion of glucose to energy necessary for mitosis (Bullough, 1954, 1955). The present author (Gelfant, 1960d) has once again challenged Bullough's theories drawn from studies on mouse ear epidermis jiz vitro. Our analysis of the effects of hormones that inhibit mitosis is shown in Table 111. Experiment 2 shows that trace quantities of metals are capable of inhibiting epidermal mitotic activity. Since most commercial hormone preparations contain some kind of metal contaminant, the question arises whether the effects of hormones in z1itr.o

25

INHIBITION OF CELL DIVISION

are even due to the hormonal moiety of the preparation used. Second, and with regard to the crucial role of the glucokinase reaction, we tested the effects of insulin and of growth hormone in the presence of a variety of hexose and pentose carbohydrate substrates, as is shown in experiment 3. The results demonstrate that there is no selective action occurring in the presence of glucose with either of these hormones. On the basis of experiments such as these, it was concluded (Gelfant, 1960d) that the mitotic effects of hormones on mouse ear epidermis in vitro are neither related to the known biological function of the hormone nor to any specific energy requirements for mitosis.

VI. Effects of Various Inhibitors on Mitosis in Vivo

A. MOUSEBODYSKIN EPIDERhlIS As explained previously (Section III,B), plucking the hairs from mouse skin provides a population of dividing epidermal cells in vivo. This system was METAPHASE INHIBITION O F

TABLE IV MITOSIS IN MOUSE

Inhibitors tested' (cone. = 0.5 mg.: period = 6 hours) None (saline) Colchicine (0.1 111s.) Podophyllin Vincaleukoblastine Chloral hydrate Maleuric acid Mercaptoethanol Nitrogen mustard Aminopterin Aminopterin (30 niin.) Aminopterin (0.5 mg./day/3 days) colchicine (0.1 mg. - 6 hours)

+

BODY SK I N EPIDERMIS in ?'i?;O" No. mitoses (metaphase) per cni. plucked epictermis" _1.9 21.7 55.9 362.0 1.9 1.7

2.9 0.9 1.1 0.5 3.0

" Effects of various inhibitors compared with colchicine. Three adult male (C57BL) mice were used to test each inhibitor. Inhibitors were injected 48 hours after the epidermis was stimulated by hair plucking. Each figure represents the average number of mitoses (arrested in metaphase) per cm. unit length of epidermis overlying the area of plucked follicles in 3 animals.

used to further evaluate the metaphase inhibitory effects of compounds that had already been tested itz vitro, as shown in Table IV. The results in Table IV corroborate the it2 ~ i t r ofindings that of all the inhibitors tested only colchicine, podophyllin, and vincaleukoblastine (VLB) are capable of inhibiting mitosis in metaphase. Chloral hydrate, maleuric acid, mercaptoethanol, nitrogen mustard, and aminopterin are once again completely ineffective as metaphase inhibitors - this time iiz ~ i v o .The fact that VLB was more effective than podophyllin and particularly colchicine in arresting rnetaphases may be due in part

26

SEYMOUR GELFANT

to the quantity of inhibitors injected; it may also be that VLB is a more efficient metaphase inhibitor than colchicine in this system as it is in Ehrlich ascites tumor cells (Cutts, 1961) and in bone marrow cells of the Syrian hamster (Cardinali et d., 1961). The experiments with aminopterin in Table IV require special comment. In view of the reports by Jacobson (personal communication and publications; Jacobson and Cathie, 1960a,b) that aminopterin loses its metaphase inhibitory effects within 24 hours in vitro due to inactivation, we tested aminopterin for a short period of 30 minutes, in addition to the regular 6-hour period as shown in Table IV. The results are clear. Aminopterin does not inhibit epidermal mitosis in metaphase either irt vivo after 30 minutes or 6 hours, or in vitro (Table I) after 1 hour (not listed in the table) or 4 hours of exposure. Thus we have been unable to confirm Jacobson's claims regarding the metaphaseinhibitory actions of aminopterin that occur within 15 minutes in hanging-drop cultures of various tissues. There is, however, a good indication, as shown by the last experiment in Table IV, that aminopterin inhibits cell division in interphdse in mouse skin epidermis in vivo. When aminopterin is administered over a 3-day period as a method of producing a general folic acid-vitamin deficiency, stimulated epidermal cells do not enter mitosis, as seen by the relatively few cells arrested in metaphase by colchicine.

B.

RAT

UTERUS

The experiments in Table V emphasize the point that the inhibitory effects of aminopterin are indeed mediated through the production of a folic acid-folinic acid-vitamin deficiency. Continuous exposure to aminopterin over a 4-day period blocks cell division in interphase, and most of the estrogen-stimulated rat uterine celis do not enter mitosis to be arrested by colchicine. The metabolic mechanism of action of aminopterin inhibition of cell division is demonstrated by the simultaneous administration of folinic acid (citrovorum factor or CF). Folinic acid bypasses the metabolic point of action of aminopterin, and now estrogenTABLE V EFFECTSOF AMINOPTERIN AND NITROGEN MUSTARD ON MITOTIC ACTIVITY I N RAT UTERUS in Vivo" Experimental treatment (per day, 3-4 days)

Average no. mitosesb all uterine tissues

Estrogen Estrogen nitrogen mustard Estrogen f aminopterin Estrogen aminopterin (CF) Castrate control

+ +

+

32.2 1.4 6.5 39.2 0.1

* Adapted from Gelfant et al. ( 1 9 5 5 ) . ' Mitoses developing over a 6-hour period were arrested in metaphase by colchicine. Citrovorum factor (folinic acid).

INHIBITION OF CELL DIVISION

27

stimulated cells are free to leave interphase and divide. Although these experiments do not elucidate the particular interphase event that is blocked by aminopterin, they do once again refute the contention of any generalized or significant inhibition of metaphase by the action of this compound. Table V also demonstrates the stage of cell division inhibited by nitrogen mustard. The previous experiments on mouse epidermis in vitro and in vivo (Tables I and IV) showed that nitrogen mustard was not a metaphase inhibitor. The present experiment on rat uterus shows that this compound is an interphase inhibitor. Like aminopterin, continuous exposure to nitrogen mustard blocks cell division in interphase, and estrogen-stimulated rat uterine cells cannot enter mitosis to be arrested by colchicine. These results also settle the issue regarding any significant inhibition of metaphase by the action of nitrogen mustard. VII. Effects of Various Inhibitors on DNA Synthesis in Vitro and

in Vivo The foIIowing experiments were designed to evaluate the effects of the inhibitors of DNA synthesis. itz Vitro A. MOUSEEAR EPIDERMIS

Five adult male mice were used. Each figure in Table VI represents the average number of labeled interphase nuclei or metaphase figures per cm. unit length of epidermis in 5 ear fragments incubated for 4 hours at 3 8 O C . in a saline medium containing glucose and 5 pc. tritiated thymidine ( thymidine-H3).

in Vim B. MOUSEBODYSKINEPIDERMIS Experirneitt I. Three adult male mice were used to test each inhibitor. Inhibitors were injected 48 hours after the epidermis was stimulated by hair plucking. Thymidine-H3 was injected 30 minutes later. The animals were killed 6 hours after injection of inhibitors with one exception: one group received aminopterin for 3 days and thymidine-H3 was injected S l / , hours before the animals were killed. Each figure in Table VI represents the average number of labeled interphase nuclei per cm. unit length of epidermis overlying the area of plucked hair follicles. Experiment 2. Three adult male mice were used to test each inhibitor. Body skin was wounded by cutting. Inhibitors were injected immediately and each day for 3 days. Thymidine-H3 was injected 45 minutes before the animals were killed (54 hours after wounding). Each figure in Table VI represents the average number of labeled interphase nuclei per mm. area of epidermis adjacent to the wound. For details of the autoradiographic procedures see Gelfant (1962). The experiments in Table VI were primarily designed to examine various reports that maleuric acid, nitrogen mustard, and aminopterin prevent mitosis

28

SEYMOUR GELFANT

by inhibiting DNA synthesis (see discussion and references in Section IV). DNA synthesis was studied by autoradiographic determination of tritium labeled thymidine incorporation into DNA of individual interphase nuclei. The results of these experiments carried out both iiz vitvo and in viz~oand in a number of TABLE V I INHIBITORS ON MITOSISAND EFFECTSOF VARIOUS DNA SYNTHESIS in ilitro AND in i ~ i t w ~

ON

Mouse Ear Epidermis in Vi1r.o

Inhibitors tested in z h v (0.01 mM)

No. labeled interphase nuclei

No. mitoses (metaphase)

41.2 89.8 19.8 26.2 33.0 39.8 49.0 64.0

0.5 9.0 5 .O 8.7 0.2 0.1 0.3 9.6

None Colchicine Podophyll in VLB (0.002 mM) Maleuric acid ( M A ) Nitrogen mustard Aminopterin Colchicine MA

+

Mouse Body Skin Epidermis in V i m

No. labeled interphase nuclei: Epidermis stimulated by Inhibitors tested

it1

rlirmo

Experiment 1*: Hair plucking

Experiment 2‘: Wounding

72.6 146.3 144.7 144.7 108.3 201.3 83.7

176.0 184.3 172.0

None (saline) Colchicine Podophyllin Vincaleukoblastine Chloral hydrate Maleuric Acid Mercaptoethanol Actidione N i trogrn mustard Aminopterin Aminopterin ( 3 days, Expt. 1)

-

172.3 145.6 128.6

-

156.6 150.3 154.3 160.0 170.3

-

” See text for description of experiments.

’Expt. 1 (0.5 mg.-6

hour period). Expt. 2 (0.5 mg./day/3 days).

experimental systems show that there is no consistent or selective inhibitory action on DNA synthesis by any of the compounds tested. The only positive result worth noting was the apparent increase in the number of labeled interphase nuclei produced by colchicine and possibly by some of the other compounds tested. Thus with regard to maleuric acid, we have been unable to confirm any of the three inhibitory effects of this compound on cell division - as reported by

INHIBITION OF CELL DIVISION

29

Sisken and associates (1959) from studies on Ehrlich ascites tumor cells. In our experiments, maleuric acid does not inhibit DNA synthesis in mouse epidermis either in oitro or in oivo (Table VI); it does not inhibit the progression of cells from G, into mitosis, because the addition of colchicine shows that epidermal cells leave the G2period and enter mitosis iu vjtro (Table VI); and finally our experiments demonstrate that maleuric acid does not block mitosis in metaphase in mouse epidermis either iri vitro (Tables I and VI) or in viuo (Table IV). The failure of nitrogen mustard and of aminopterin to inhibit DNA synthesis (as measured by autoradiographic labeling), also stands in lack of confirmation of reports using various other methods for determining DNA synthesis in the presence of these compounds (see discussion and references in Section IV). However, we have established that both nitrogen mustard and aminopterin are mitotic inhibitors, and more specifically that they inhibit cell division in interphase. It is therefore only the mechanism of action of these compounds that remains open to speculation and to further experimentation. The possibility of a block during the G 2 period of interphase has already been mentioned with reference to nitrogen mustard in Section IV, and such an explanation may also apply to the inhibitory action of aminopterin. The evidence needed to support the G, block explanation would involve an experiment showing an accumulation of nuclei with double DNA contents (as determined by microspectrophotometric DNA measurements) after an appropriate period of exposure to nitrogen mustard or to aminopterin, and in an experimental system undergoing DNA synthesis and mitosis.11 Meanwhile we can only conclude that neither nitrogen mustard nor aminopterin inhibits DNA synthesis, as shown by autoradiographic analysis of labeled thymidine incorporation in mouse epidermis.

VIII. Effects of Various Inhibitors on Growth and on Mitosis iri V i m Growth may be due to an increase in the number and/or the size of cells (involving protoplasmic or dry mass increase). The experiments in Table VII dissociate growth by cell division from growth by cell enlargement by selectively inhibiting cell division in the estrogen-stimulated rat uterus. Thus one can study either of the two major aspects of growth, in relation to each other and in relation to total uterine growth. In addition, the experiments in Table VII present a controlled comparison of the inhibitory effects of mitotic inhibitors in a variety of different uterine cell types. The generalized nature of the inhibitory actions of nitrogen mustard and of aminopterin in interphase can be seen in all uterine tissues. Epithelial, gland, connective tissue, and muscle cells are blocked in interphase. This is demonstrated by the fact that they cannot enter mitosis to be arrested by colchicine. Folinic 1 1 Such evidence has recently been provided for nitrogen mustard in mouse fibroblasts grown in tsitro (Brewer et a[.. 1961).

30

SEYMOUR GELFANT

acid (CF) effectively reverses aminopterin inhibition of cell division in all tissues, and these cells are then capable of entering mitosis. Second, there is a direct correlation between mitotic activity and total uterine growth. Inhibition of cell division, or reversal, is reflected in comparable changes in uterine dry weight, illustrating the relationships of both cell division and cell enlargement to total growth. TABLE VII EFFECTSOF AMINOPTERIN, NITROGEN MUSTARD, AND COLCHICINE ON GROWTH AND ON MITOTIC ACTIVITYIN RATUTERUS in ViUO" Experiment 1 Treatment per day 3-4 days Estrogen Estrogen H N 2 Estrogen aminopt. Estrogen aminopt. Castrate control

+ + +

+ (CF)

Uterine growth dry wt. (mg.)

Uterine mitotic activity No. mitoses each tissueb Epithelium Gland Stroma Muscle

16.4 8.0 9.0 11.4 4.6

25.2 1.8 7.5 44.0

9.6 0.7 3.3

15.4

0.0

0.1

54.6 1.2 12.9 28.2 0.2

39.2 1.8 2.4

69.0 0.0

Experiment 2 Treatment per day 3-4 days

Body wt. (P.1

Uterine growth dry wt. (ma.)

Estrogen Estrogen colchicine Inanition estrogen Castrate control

75.0 51.0 41.0 75.0

23.6 8.8 18.8 8.3

+ +

a

Adapted from Gelfant et al. (1955). Mitoses developing over a 6-hour period were arrested in metaphase by colchicine.

Since daily administration of nitrogen mustard or of aminopterin for 3 to 4 days causes inanition and loss of body weight, a control experiment was designed (experiment 2 of Table VII). The results demonstrate that inanition in itself is not responsible for the inhibition of uterine growth when mitotic inhibitors are administered, because uterine dry weight increased considerably in the estrogen-treated starved animals. The inhibitory effects on uterine growth produced by a continuous %day exposure to colchicine are particularly noteworthy because they illustrate that growth by cell division can be inhibited equally as well, whether the mitotic inhibitor blocks cell division in interphase (aminopterin, nitrogen mustard) or in metaphase (colchicine) .

IX. Comment on the Inhibition of Cell Division by X-Irradiation The antimitotic actions of X-irradiation have generally been attributed to the fact that DNA synthesis is radiosensitive. In an excellent review on the inhibition of DNA synthesis and mitosis by ionizing radiations, Abrarns (1961) concludes:

INHIBITION OF CELL DIVISION

31

“To summarize, present evidence suggests that ionizing radiations suppress mitotic division and independently interfere with D N A synthesis. For the latter action, sensitivity is greatest in the interphase period before actual synthesis begins (during the GIperiod.]” Thus, the inhibition of cell division by ionizing radiations is currently being viewed in terms of multiple effects during interphase, and also in terms of the dissociable GI,S, and G, periods of the cell division cycle. One report not covered by Abrams and particularly relevant to the present review is the recent paper by Das and Alfert (1961) showing that although X-irradiation inhibits mitosis in onion root tip cells, it actually stimulates D N A synthesis. Accelerated D N A synthesis was detected both by autoradiographic grain counts of thymidine-H3 incorporation and by microspectrophotometric measurements of D N A on the same nuclei. In this case, irradiation blocks cell division in the G2period of interphase. The synthesis of DNA, however, is not only free to continue during the block, but it is actually stimulated by irradiation. In another report Alfert and Das (1961) go one step further in this analysis by showing that thymidine incorporation in X-rayed material is stimulated at the beginning of, but gradually decreases during, the period of D N A synthesis. They suggest that irradiation initiates DNA synthesis only in unduplicated chromosome regions, but has no such effect on chromosome regions in which D N A has already duplicated. This differential behavior of D N A synthesis to X-rays further illustrates the principle of independent and dissociable events involving reactions on a precursor level as discussed in Section I1 of the present review. The situation for ionizing radiations thus closely resembles the one described for nitrogen mustard in Section IV, and it may also explain the inability to detect an inhibition of DNA synthesis with either nitrogen mustard or with aminopterin in the experiments on mouse epidermis shown in Table VI. The evidence that the mitotic block during interphase following irradiation (presumably during the G, period) is independent of any influence on D N A synthesis, and, the possibility that DNA synthesis may either be inhibited (if the cell is in G I ) , that it may continue, or may even be accelerated (if the cell is in early, middle, or late S period) provides a new insight into the problem of the inhibition of cell division. And if we employ the radiomimetic concept (that certain chemicals act like ionizing radiations on cells), this insight can also be used profitably to investigate the mode of action of chemical inhibitors.

X. Comment o n Cancer Chemotherapy Screening Methods A comment on cancer chemotherapy studies seems appropriate in view of the subject of the present review, and because almost all of the compounds used or discussed in the present review have either been screened as antitumor agents or bear some practical relationship to the cancer problem. A valuable description of the various programs on antitumor chemotherapeutic screening and drug evalua-

32

SEYMOUR GELFANT

tion may be found in the recent review by Goldin et al. (1961), and in the series of supplements to Caizcer Research (for example, Supplements IX-XII, 1961). The point to be made here can best be illustrated by the experience in the present review with vincaleukoblastine (VLB), one of the drugs currently in focus as a cure for cancer (see Canadian Cancer Conference, 1961). The reader is referred to the discussion of this compound in Section IV, and to the experimental results obtained with VLB in Tables I, IV, and VI. It was clear from the beginning that the alkaloid vincaleukoblastine is a metaphase inhibitor of mitosis, and it seems to be more efficient than colchicine, another alkaloid-metaphase inhibitor. It was also clear that VLB would not demonstrate any preferential action on tumor cells because it effectively arrested mitosis in metaphase in normal mouse epidermis both in vitro and in vivo. Furthermore, it is also clear that a cell cannot remain detained in metaphase indefinitely; it must either overcome the block and divide or it will degenerate; and this is true for both normal and neoplastic cells. Although VLB was not tested in our experiments on rat uterus iit vivo (Table VII), it was shown that total growth, as measured by the weight of an organ, can be inhibited or reduced by mitotic inhibitors that block cell division in metaphase. Thus the various laboratory and clinical studies dealing with the effects of VLB on tumor growths have probably demonstrated nothing more than a relathe inhibition or reduction of a fast growing tumor system as compared with slower growing normal tissues. The so-called “remissions” produced by VLB (increased survival time of tumor bearing animals or patients, decreased organ and tumor mass size, and marked improvement in general clinical condition) have almost always been partial remissions and of relatively short duration - probably until a sufficient number of normal cells crucial for survival were inactivated by VLB. In view of the above discussion, and primarily on the basis that vincaleukoblastine is a general metaphase inhibitor of cell division, it seems highly unlikely that this compound will have any profound impact as a cure for cancer. Consequently, unless its oncolytic action has nothing to do with metaphase arrest, the final evaluation of VLB as a cancer cure will probably turn out to be quite similar to the clinical results obtained with another alkaloid and general metaphase inhibitor, namely, colchicine (Eigsti and Dustin, 1955). Thus the real problem underlying the chemotherapy of neoplastic growth and cell division, whether it be genetic, epigenetic, viral, or spontaneously induced (Huxley, 1956, 1957), involves an understanding and an active appreciation of the process of cell division. XI. Concluding Remarks One final point to be considered involves the distinction between the significance of a mitotic inhibitor as an experimental tool to study the process of cell division, and the significance of the same inhibitor in terms of its generalized usefulness in all experimental systems. In the opinion of the present author, it

INHIBITION OF CELL DIVISION

33

should be stated that even though the effects of an inhibitor may not be generalized, it does not invalidate or minimize the importance of the inhibitor as an analytical tool for studying cell division in the experimental system in which it works. For example, one of the illustrations cited in Section I1 (Mazia et d., 1960) involved a brilliant and profound analysis of an essential mechanism of cell division using the sulfhydryl-containing compound, mercaptoethanol. This compound was used to block mitosis in metaphase in echinoderm eggs in order to study the mechanism of reproduction of the mitotic centers. In the introductory remarks to this paper, Mazia emphasizes the fact “that mercaptoethanol is being used here solely as an analytical tool. In the following discussion, no part of the argument depends on any stipulation as to the mechanism of action of mercaptoethanol . . . Nor is there any reason to affirm or to doubt that other and even related chemicals might be used for the same purpose.” In this case the authors are not even concerned with the mechanism of action of the inhibitor, let alone with its generalized usefulness in other experimental systems. So that even though mercaptoethanol does not inhibit mitosis in metaphase in adult mammalian cells in r*i/ro or in vivo, this in no way depreciates from its value as an experimental tool to inhibit metaphase in dividing echinoderm eggs. The same situation applies to chloral hydrate. W e have established (Section V,A) the fact that chloral hydrate is not a generalized spindle or metaphase inhibitor when its actions are compared with colchicine, VLB, or podophyllin on mouse epidermis both itz riti.0 and itz vivo. Nevertheless, chloral hydrate has been used with tremendous advantage in grasshopper spermatocytes by Ris (1949) to elucidate the mechanisms involved in anaphase movement. In this system chloral hydrate functions as a precise experimental tool to dissect and to dissociate the two processes involved in the spindle movement of chromosomes during mitosis. There are a number of reasons why inhibitors may not have or may not appear to have the same effects on cell division in different experimental systems. To begin with, as Ludford (1953) points out, a specific influence on cell division produced by injecting a compound into the mammalian body is quite “a different matter from deranging mitosis by adding chemicals to sea water containing the eggs of marine animals, or to water in which the roots of plants are grown, or to the medium used for growing tissues [or microorganisms] in cultures.” In addition, even if all conditions were optimal, i.e., if application and specificity of action of the inhibitor were successful, and the investigator had a thorough understanding of both the experimental material and the process of cell division, we would still have to contend with the problems of inherent metabolic and physiological differences that exist in different types of cells (many egg cells, for example, contain nutrient stores and DNA degradation products in th, cytoplasm), and also with the problem of different cell populations that exist within a presumably homogeneous experimental system [as in mouse epidermis

34

SEYMOUR GELFANT

(Gelfant, 1 9 6 2 ) ] . Finally, and superimposed upon all of these conditions, is the question of multiple effects of inhibitors under various experimental and physiological conditions. Yet in spite of all these complexities, it is still possible to establish a predictable, significant, and generalized evaluation of the mechanism of action of a mitotic inhibitor. This can be done because the basic core of biochemical and physiological events that governs the process of cell division is essentially the same in all cell types. What is required is an understanding of the experimental system and a pattern of investigation that focuses on the proem of cell division. For inhibition and progress are indeed the same problem.

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