- Email: [email protected]
Anomalous cytoplasmic vacuoles and cell division
564
Experimental
ANOMALOUS
CYTOPLASMIC
Cell Research
VACUOLES
AND
21, 564-568
(1960)
CELL
DIVISION1 S. FEDOROFF Department
OJ Anatomy, Received
University December
of Saskatchewan,
Canada
21, 1959
A NOM~LOI'S.
cells \vere observed in cultures of strain L cells through numerous subcultivations for more than one year. The anomalous cells contained one or more large vacuoles in the cytoplasm. The cytoplasmic anomaly seemed not to interfere with division of the cells. To my knowledge, such changes in strain L cells have not been reported and therefore they are described herewith.
METHODS
AND
MATERIALS
In August of 1955 a flask of strain L cells [I, 61, designated A929-217-108, was obtained from Dr. W. R. Earle. For the first four months these cells were grown in a medium consisting of 40 per cent horse serum, 50 per cent balanced salt solution and 10 per cent chick embryonic extract, but at the end of the fourth month the medium was changed to 30 per cent horse serum, 50 per cent balanced salt solution and 20 per cent synthetic medium 199 [5], which later was replaced by synthetic medium CMRL-i066.2 The cultures were routinely incubated at 37°C. The medium was changed completely twice a week, and the cultures were subcultured every 8-10 days. RESULTS
Some vacuolated cells were observed among healthy strain L cells in one culture. At first these cells were thought to be degenerating. However, after the culture was washed and new medium added, the vacuolated cells persisted. After similar treatment other degenerating cells usually either die or lose their vacuoles, but in the present case, va cuolated cells kvere present even after numerous suhcultivations (Fig. 1). These cells have now been 1 This work was presented at the First Pan-American Cancer Cytology Congress, Miami, Fla., April 25-29, 1957. It was done while working under a grant (607-7-15) from the Department of National Health and Welfare, Canada and a grant from the Leukemia Society Inc., New York. 2 Obtained from the Connaught Medical Research Laboratories, University of Toronto. The medium is a modification of formula 858(4). Experimental
Cell
Research
21
Anomalous
cyfoplasmic
565
vacuoles and cell division
carried by serial transplantation for more than one year. The number of vacuolated cells in the cultures varied from approximately 2 per cent to 20 per cent of the cells. The fact that these vacuolated cells persisted in the culcultures seems to indicate that they were not merely the usual type of degen-
Fig.
l.--Anomalous
vacuolated
cells in a culture
of strain
L cells.
Unstained,
unfixed
( x 364).
erating cells. It should be emphasized that despite identical culturing conditions, this type of cell was not observed either in other flasks of strain I, cells which originated from the culture received in 1955, or in a substrain of strain I, cells derived from a culture received from Dr. Earle at an earlier date (1953). 1Ioreover, the alteration was not observed in other cell strains (HeLa, Chang’s human kidney and Chang’s conjunctiva) maintained in this lahoratory. Fixed and stained covcrslip preparations were studied microscopically. It was seen that many of the vacuolated cells were dividing mitotically, which explains their presence in the cultures in more or less constant numbers (Fig. 2 o, b, c, d). When a number of small vacuoles were present in the cytoplasm of a dividing cell, the vacuoles segregated at random and both daughter cells received some of the vacuoles (Fig. 26). If only one vacuole was present, it was found in one of the daughter cells while the other had Experimental
Cell Research
21
566
S. Fedoroff
none (Fig. 2c, d). It seemed that the size of the vacuole had no influence on nuclear division, but that it did influence cptoplasmic division. Thus, if very large vacuoles were present in the cytoplasm of the cell, the nucleus but not the cytoplasm divided, and as a result, a binucleated cell was formed (Fig. 3 a).
e Fig. Z.-Dividing vacuolated strain L cells. (b) cell with multiple vacuoles in anaphase ( x 2619); (d) telophase ( x 2490).
Bouin’s fixative. H. & E. (a) metaphase ( x 2064); ( x 2419); (c) cell with single vacuole iu anaphase
Occasionally vacuolated cells containing three, four or more nuclei were observed (Fig. 3b). One might expect that under such bizarre cytoplasmic conditions abnormal nuclear divisions would be found, but no gross abnormalities mere observed.
Anomalous
cytoplasmic
567
vacuoles and cell division
After Bouin’s fixation, the vacuolar content did not stain with hematoxylin or with eosin. It was negative for desoxyribonucleic acid when tested by the Feulgen reaction, and negative for polpsaccharides when tested by the periodic acid-Schift’ reaction. The vacuoles were unstainable vvith Sudan IV, Sudan Black and osmic acid, indicating that lipids were probably not present.
Fig. 3.-Multinucleated in early prophase
vacuolated strain L cells. Bouin’s fixative. H. Lyi E. (a) binucleatetl ( x 2361); (b) multivacuolated cell with three dividing nuclei ( Y 2582).
cell
When .Janus green was added to the medium, mitochondria were stained, but the vacuolar content was not. The material in the vacuoles stained brilliantly when neutral red was added to the medium.
DISCUSSION
Geg [:I] observed vacuoles in IZhrlich mouse c.arcinoma cells which had been cultured for three years. The vacuolated cells survived numerous transplantations and even an intraperitoneal passage. Gey suggested that these cellular changes might be due to the presence of a “viral contaminant or passenger agent”. It is possible that the cellular abnormality described in Experimenfal
Cell
Research
21
568
S. Fedoroff
the present paper might also be due to a virus, but there is no direct evidence at present to support this hypothesis. Vacuolated cells were also observed by Fjelde [a] in cultures of strain S (sarcoma) cells. These cells were the predominant type and were constantly present in cultures of this strain. The content of the vacuoles was tested for lipids, polysaccharides, pentosenucleic acid, desoxynucleic acid, and argcninc and all tests were negative. The importance of the observations reported in the present paper seems to lie in the fact that in spite of the large vacuoles, mitoses were not disturbed. If we assume that the vacuoles are an expression of a pathologic process in the cells, it seems unusual that mitoses should not be aflected. Vice versa, if the vacuoles are the expression of a functional state, it seems equally unusual that a highly active cell should be dividing mitotically.
SUMMARY
Some cells in a culture of strain 1, cells contained large vacuoles, which were present even during mitotic division. During numerous subcultirations for a period of more than one year this phenomenon did not disappear. Nuclear division seemed not to be affected regardless of the size or number of the vacuoles, but cytoplasmic division appeared to be inhibited when very large vacuoles were present. The vacuolar content did not stain with Sudan I\‘, Sudan Black or osmic acid, which indicates that it was not of lipid nature. The vacuolar material was also unstainable by the Feulgen and periodic acitl-Schiff methods, indicating that dcsoxyribonucleic acid and polysaccharides were absent. On the other hand, it stained readily with neutral red. REFERENCES 1. 2. 3. 4. 5. 6.
EARLE,
R., J. Nat!. Cancer Inst. 4, 165 (1943). Cancer 8, 845 (1955). GEY. G. 0.. Harveu Lectures 1954-1955, D. 154 (1956). HEAVY, G. ‘M., IT&R, I). C. and PARK&R, R. iI., &x. Sot. Exptl. Biol. Med. 89, 71 (1955). MORGAN, .J. E’., MORTON, H. J. and PARKER, R. C.. Proc. Sot. Exptl. Bio!. Med. 73 (1950). S.~FORD, K. I<., Ennm, W. R. and LIKELY, J. D., .I. K&l. Cancer Inst. 9, 229 (1948). FJELDE,
Experimental
W.
A.,
Cell
Research
21
Experimental
ANOMALOUS
CYTOPLASMIC
Cell Research
VACUOLES
AND
21, 564-568
(1960)
CELL
DIVISION1 S. FEDOROFF Department
OJ Anatomy, Received
University December
of Saskatchewan,
Canada
21, 1959
A NOM~LOI'S.
cells \vere observed in cultures of strain L cells through numerous subcultivations for more than one year. The anomalous cells contained one or more large vacuoles in the cytoplasm. The cytoplasmic anomaly seemed not to interfere with division of the cells. To my knowledge, such changes in strain L cells have not been reported and therefore they are described herewith.
METHODS
AND
MATERIALS
In August of 1955 a flask of strain L cells [I, 61, designated A929-217-108, was obtained from Dr. W. R. Earle. For the first four months these cells were grown in a medium consisting of 40 per cent horse serum, 50 per cent balanced salt solution and 10 per cent chick embryonic extract, but at the end of the fourth month the medium was changed to 30 per cent horse serum, 50 per cent balanced salt solution and 20 per cent synthetic medium 199 [5], which later was replaced by synthetic medium CMRL-i066.2 The cultures were routinely incubated at 37°C. The medium was changed completely twice a week, and the cultures were subcultured every 8-10 days. RESULTS
Some vacuolated cells were observed among healthy strain L cells in one culture. At first these cells were thought to be degenerating. However, after the culture was washed and new medium added, the vacuolated cells persisted. After similar treatment other degenerating cells usually either die or lose their vacuoles, but in the present case, va cuolated cells kvere present even after numerous suhcultivations (Fig. 1). These cells have now been 1 This work was presented at the First Pan-American Cancer Cytology Congress, Miami, Fla., April 25-29, 1957. It was done while working under a grant (607-7-15) from the Department of National Health and Welfare, Canada and a grant from the Leukemia Society Inc., New York. 2 Obtained from the Connaught Medical Research Laboratories, University of Toronto. The medium is a modification of formula 858(4). Experimental
Cell
Research
21
Anomalous
cyfoplasmic
565
vacuoles and cell division
carried by serial transplantation for more than one year. The number of vacuolated cells in the cultures varied from approximately 2 per cent to 20 per cent of the cells. The fact that these vacuolated cells persisted in the culcultures seems to indicate that they were not merely the usual type of degen-
Fig.
l.--Anomalous
vacuolated
cells in a culture
of strain
L cells.
Unstained,
unfixed
( x 364).
erating cells. It should be emphasized that despite identical culturing conditions, this type of cell was not observed either in other flasks of strain I, cells which originated from the culture received in 1955, or in a substrain of strain I, cells derived from a culture received from Dr. Earle at an earlier date (1953). 1Ioreover, the alteration was not observed in other cell strains (HeLa, Chang’s human kidney and Chang’s conjunctiva) maintained in this lahoratory. Fixed and stained covcrslip preparations were studied microscopically. It was seen that many of the vacuolated cells were dividing mitotically, which explains their presence in the cultures in more or less constant numbers (Fig. 2 o, b, c, d). When a number of small vacuoles were present in the cytoplasm of a dividing cell, the vacuoles segregated at random and both daughter cells received some of the vacuoles (Fig. 26). If only one vacuole was present, it was found in one of the daughter cells while the other had Experimental
Cell Research
21
566
S. Fedoroff
none (Fig. 2c, d). It seemed that the size of the vacuole had no influence on nuclear division, but that it did influence cptoplasmic division. Thus, if very large vacuoles were present in the cytoplasm of the cell, the nucleus but not the cytoplasm divided, and as a result, a binucleated cell was formed (Fig. 3 a).
e Fig. Z.-Dividing vacuolated strain L cells. (b) cell with multiple vacuoles in anaphase ( x 2619); (d) telophase ( x 2490).
Bouin’s fixative. H. & E. (a) metaphase ( x 2064); ( x 2419); (c) cell with single vacuole iu anaphase
Occasionally vacuolated cells containing three, four or more nuclei were observed (Fig. 3b). One might expect that under such bizarre cytoplasmic conditions abnormal nuclear divisions would be found, but no gross abnormalities mere observed.
Anomalous
cytoplasmic
567
vacuoles and cell division
After Bouin’s fixation, the vacuolar content did not stain with hematoxylin or with eosin. It was negative for desoxyribonucleic acid when tested by the Feulgen reaction, and negative for polpsaccharides when tested by the periodic acid-Schift’ reaction. The vacuoles were unstainable vvith Sudan IV, Sudan Black and osmic acid, indicating that lipids were probably not present.
Fig. 3.-Multinucleated in early prophase
vacuolated strain L cells. Bouin’s fixative. H. Lyi E. (a) binucleatetl ( x 2361); (b) multivacuolated cell with three dividing nuclei ( Y 2582).
cell
When .Janus green was added to the medium, mitochondria were stained, but the vacuolar content was not. The material in the vacuoles stained brilliantly when neutral red was added to the medium.
DISCUSSION
Geg [:I] observed vacuoles in IZhrlich mouse c.arcinoma cells which had been cultured for three years. The vacuolated cells survived numerous transplantations and even an intraperitoneal passage. Gey suggested that these cellular changes might be due to the presence of a “viral contaminant or passenger agent”. It is possible that the cellular abnormality described in Experimenfal
Cell
Research
21
568
S. Fedoroff
the present paper might also be due to a virus, but there is no direct evidence at present to support this hypothesis. Vacuolated cells were also observed by Fjelde [a] in cultures of strain S (sarcoma) cells. These cells were the predominant type and were constantly present in cultures of this strain. The content of the vacuoles was tested for lipids, polysaccharides, pentosenucleic acid, desoxynucleic acid, and argcninc and all tests were negative. The importance of the observations reported in the present paper seems to lie in the fact that in spite of the large vacuoles, mitoses were not disturbed. If we assume that the vacuoles are an expression of a pathologic process in the cells, it seems unusual that mitoses should not be aflected. Vice versa, if the vacuoles are the expression of a functional state, it seems equally unusual that a highly active cell should be dividing mitotically.
SUMMARY
Some cells in a culture of strain 1, cells contained large vacuoles, which were present even during mitotic division. During numerous subcultirations for a period of more than one year this phenomenon did not disappear. Nuclear division seemed not to be affected regardless of the size or number of the vacuoles, but cytoplasmic division appeared to be inhibited when very large vacuoles were present. The vacuolar content did not stain with Sudan I\‘, Sudan Black or osmic acid, which indicates that it was not of lipid nature. The vacuolar material was also unstainable by the Feulgen and periodic acitl-Schiff methods, indicating that dcsoxyribonucleic acid and polysaccharides were absent. On the other hand, it stained readily with neutral red. REFERENCES 1. 2. 3. 4. 5. 6.
EARLE,
R., J. Nat!. Cancer Inst. 4, 165 (1943). Cancer 8, 845 (1955). GEY. G. 0.. Harveu Lectures 1954-1955, D. 154 (1956). HEAVY, G. ‘M., IT&R, I). C. and PARK&R, R. iI., &x. Sot. Exptl. Biol. Med. 89, 71 (1955). MORGAN, .J. E’., MORTON, H. J. and PARKER, R. C.. Proc. Sot. Exptl. Bio!. Med. 73 (1950). S.~FORD, K. I<., Ennm, W. R. and LIKELY, J. D., .I. K&l. Cancer Inst. 9, 229 (1948). FJELDE,
Experimental
W.
A.,
Cell
Research
21