66. The Cycle o f Tumor Cells in a Transplant Generation o f the Yoshida Sarcoma.

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1 No. 6.] The Cycle o f Tumor Cells in a Transplant Generation o f the Yoshida Sarcoma. By Sajiro MAKINO. Zoological Institute, Hokkaido University. (Comm. by T. KOMA1, M.J.A., June 12, 1951.) The Yoshida sarcoma is a kind of ascite sarcoma which grows in the peritoneal cavity of white rats (Rattus norvegiens) in a form of malignant tumor. According to Yoshida (1949, Gann 40), the sarcoma originally developed in an albino rat which had been fed with o-amidoazotoluene for 3 months and then applied cutaneously with potassium arsenite solution for about 3 months. The tumor ascites are of milky appearance, and 1 cc of the ascites contains approximately 1 million tumor cells, which occur in a form of suspension. The successive transmission is made from rat to rat by intraperitoneal introduction of a droplet of the tumor ascites. The tumor cells rapidly proliferate in a new host after transplantation, bringing about enormous increase of ascites, and the diseased animal dies in 12 days on the average. The Yoshida sarcoma. has many advantages for cytological investigation. Morphological observations and statistical survey on the mitotic abnormalities have been carried out. Furthermore, the mitotic rate, the variation in chromosome number and morphology in the tumor cells have been studied. From these studies the following conclusion has been drawn : -- There is a strain of tumor cells which are primarily responsible for the formation of the tumor. These cells are characterized by having a well-balanced subdiploid set of chromosomes, 40 or thereabout in number. They multiply, displaying quite normal mitotic behavior (Figs. 9-12). The chromosome complex of these cells is highly remarkable in having a characteristic constitution, with two distinct chromosome groups which are apparently dissimilar in nature (Figs. 2--4). One of the groups consists of rod-shaped elements ranging from 22 to 24 in number, while the other group comprises V-shaped elements of varying sizes, from 16 to 18 in number. It is an established fact that the normal somatic cell of the white rat contains 42 chromosomes and all of them are rodshaped (Fig. 1) ; in no case has any V-shaped element been observed. Thus, the chromosome set of the tumor cell shows a dis- 1) Contribution Hokkaido University, No. 253 from the Sapporo, Japan. Zoological Institute, Faculty of Science,

2 288 S. MAKING. Vol. 27, tinct morphological difference cell (Figs. 5-8). Undoubtedly, from that of the rod-shaped the ordinary somatic chromosomes, about Fig. 1, Chromosomes of a normal liver cell, 42 chroms. Figs. 2-4, chromosomes of Yoshida sarcoma cells, 40, 41 and 39 chroms. respectively. Figs. 5-8, serial alignments of chromosomes. Fig. 5, from a liver cell. Figs. 6-8, from Yoshida sarcoma cells. ca x in pair, occurring in the tumor cell, correspond to certain 12 pairs of the chromosomes found in somatic cell of the host, because of their morphological likeness. In other words, these rod-shaped elements in the tumor cell seem to be derived from the ordinary tissue cell of the white rat. As for the set of V-shaped chromosomes, on the contrary, one fails to recognize any comparable element in the ordinary cell of the rat, since the latter cell contains no chromosome resembling the V-shaped element found in the tumor cell. This indicates that the V-chromosomes can be regarded as proper to the tumor cell. However, nothing is known about the origin and nature of these V-shaped chromosomes. At any rate, on account of this characteristic, the chromosome set of the tumor cell shows a marked contrast to that of the host tissue (compare Fig. 5 with Figs. 6-8). So far as my observations go, no transitional type of chromosomes between normal cells and tumor cells has been detected (cf. Makino 1951, Gann 42). The individuality of the chromosomes in the strain of the tumor cell remains unaltered through successive transplant generations from host to host,- probably from their original ancestor, maintaining tie malignancy as well as the inherited capacity for autonomous growth. It is these tumor cells which are primarily responsible for the growth of the tumor through their regular mitotic multiplication.

3 No. 6.1 The Cycle of Tumor Cells in the Yoshida sarcoma. 289 The behavior of the tumor cells in a transplant generation cycle is as follows : - Following the transplantation of _ the tumor, a large proportion of the tumor cells introduced into the peritoneal cavity of the new host seem to undergo degeneration, because there appear many cells undergoing disintegration as well as those manifesting various abnormalities. The dividing cells are very few. The majority of the degenerating cells are large in size, and are characterized by a considerable amount of cytoplasm with a large bilobed, kidneyshaped, or lobulated nucleus.. Giant cells containing a huge nucleus Figs. 9-12, regular behavior of chromosomes during mitotic division in the strain cells of Yoshida sarcoma. 9, prophase. 10, anaphase. 11, telophase. 12, division of cell body. x Figs , some of the mitotic abnormalities found in Yoshida sarcoma cells. 13, disintegration of chromosomes at metaphase showing vacuolization. 14, irregular swelling of chroms. 15, lagging of chroms. at anaphase. 16, chromosome brigdes.17, irregular arrangement of chroms. at metaphase. 18, probably tripolar spindle. 19, tripolar division. 20, octopolar division. 21, giant cells showing more than 200 chroms. 22, a giant cell containing 3 nuclei. Fig. 23, two strain cells at resting state, from the host just after death , x , x , x , x 5T0. or many nuclei are also rather common. Obviously, these cells are derivatives of the strain cells, which have received the aberrant chromosome set through abnormal mitoses. The mitotic figures of tumor cells begin to appear in about 24 hours after transplantation. It is noteworthy that most of the

4 290 S. MAKIN0. [vol. 27, cells which are undergoing division are small-sized, and contain a characteristic chromosome set. So it is beyond question that they are the strain cells. The mitotic cells rapidly increase with time. On the 3rd or 4th day after transplantation, the strain cells undergo very active multiplication. Probably, the peritoneal fluid of the host at this stage serves as a favorable culture medium for muftin of these cells. Towards the middle part of the life span plication of the tumor-bearing animal, the cells undergoing mitosis attain the highest frequency of the whole transplant generation. Then. they gradually decrease towards the latter part of the life span of the host. With the decrease of the cells in mitosis, the frequency of cells showing mitotic abnormalities and in the course of disintegration rises. On the last day of the life of the tumor-bearing animal, mitotic figures of the strain cells abruptly decrease, while the cells showing abnormalities as well as the cells in the course of disintegratioi increase at a strikingly high rate. Mingled with these degenerating cells, there occur in the tumor ascites a number of apparently resting cells which are characterized by their small size containing small amount of cytoplasm in a condensed state, and by compact nuclei. These resting cells correspond in all probability to the strain cells, which fact has been corroborated by examination on permeability. By the end of the life of the host, the accumulation of the tumor ascites reaches an enormous amount, and brings about the death of the host. As described above, active multiplication of the strain cells takes place from the early part to the middle part of a transplant generation. During this time, a part of the proliferating strain cells become abnormal through aberrant mitosis, owing probably to the breaking down of the normal spindle mechanism, or the structural change in the chromosomes, or by some other unknown causes. The cells showing mitotic abnormalities and those in the course of disintegration increase in number at the same pace with the accumulation of the tumor ascites which takes place during the latter part of the transplant generation. It is highly probable that the accumulation of the tumor ascites, which are in the greater part degenerative products of tumor cells, brings about the change in viscosity of ascites ; this change will serve to disturb the waterrelation within the tumor cell, and cause the change of normal spindle mechanism as a result of hydration and dehydration processes, thus leading to various mitotic abnormalities (Figs ). These mitotic abnormalities can be arranged into two categories, namely, i) abnormalities involving the structural alteration in chromosomes, and ii) abnormalities in spindle mechanism such as accelerated, delayed, or suppressed, or incomplete spindle formation. To the

5 No. 6.] The Cycle of Tumor Cells in the Yoshida sarcoma. 291 former type of abnormality belong stickiness and coalescence of chromosomes, abnormal swelling and vacuolization of chromosomes, deformation of chromosomes into unusual round bodies, and disintegration of spiral chromonemata. As the second group of abnormality, there appear lagging and non-disjunction of chromosomes, chromosome-bridges and irregularities in the distribution of chromosomes at anaphase, hollow metaphase, displacement and disorientation of metaphase chromosomes, abnormal orientation of metaphase spindle, the formation of restitution nucleus and multinucleate cells, and multipolar mitoses. These mitotic abnormalities will result in the variation of the chromosome number. Obviously, cells sowing such abnormalities as above are incapable of active multiplication, so that they cannot contribute to the growth of the tumor. As stated above, the chromosome complex in the Yoshida sarcoma cell has a striking difference from that in the tissue cell of the host, in that roughly one half of the chromosomes are apparently of the host origin, while the other half consists of deformed chromosomes. The individuality of chromosomes remains unaltered through successive transplant generations. This peculiar chromosome complex and the autonomous growth of the tumor tissue may be due to a kind of mutation. Possibly, in some stage in the course of the experiment with the carcinogenic reagent, a cell or cell group in the normal tissue underwent a mutational change, and has acquired the characteristics of the tumor cell, namely, the peculiar chromosome complex, autonomous developmental capacity and the capacity for successive transmission. In these respects the sarcome cell simulates a parasitic organism. I acknowledge here my indeptedness to Dr. Kan Oguma for important criticism, and also to Dr. Taku Komai for going over the manuscript for publication. Cordial thanks are also due to my co-workers, Mr. T. Yosida, Mr. T. Tanaka and Miss K. Kano for their help in many ways during the course of this study.