- Email: [email protected]
Effects of actinomycin D, amethopterin, and 5-fluro-2′-deoxyuridine on procentriole formation in Chinese hamster fibroblasts in culture
Printed in Sweden Copyright Q 1974 by Academic Press, Inc. AN rights of reproduction in any form resewed
Experimental
EFFECTS AND
Cell Research 85 (1974) 136-142
OF ACTINOMYCIN
5-FLURO-2’-DEOXYURIDINE IN
CHINESE
HAMSTER P. H. DeFOORl
The University
D, AMETHOPTERIN, ON PROCENTRIOLE
FIBROBLASTS
IN
FORMATION CULTURE
and E. STUBBLEFIELD
of Texas M. D. Anderson Hospital and Tumor Institute, and the Graduate of Biomedical Sciences, Houston, Tex. 77025, USA
School
SUMMARY Synchronized Chinese hamster fibroblasts (Don-C) were cultured for 6 h in the presence of amethopterin, 5-fluorodeoxyuridine (FUdR), or actinomycin D and were then examined by electron microscopy of thin sections for the presence or absence of daughter procentrioles. Control cells were at mid S phase and contained obvious procentrioles in about 1 profile out of 140. Treatment with FUdR or amethopterin did not reduce this frequency significantly. However, serial section analysis did demonstrate a lack of procentrioles in some cells treated with amethopterin. Actinomycin was quite effective in preventing daughter procentriole formation.
Centrioles were first observed in 1886 by Van Beneden & Boveri [l]. However, studies of the fine structure and mode of replication of these minute organelles were hindered until the advent of electron microscopy in the 1950s. Since then, many studies on centriole structure and function [2-111 have led to speculations that centrioles might be selfreplicating structures containing centriolespecific DNA. However, it has not yet been possible to discern this with certainty, and investigations of the factors involved in centriole replication are needed to further our understanding in this regard. In dividing cells, the diplosomal centrioles replicate in each cell cycle and are separated at mitosis in such a way that each newly divided cell has two centrioles [l, 10, 11, 141. 1 Current address: Department of Veterinary Microbiology, Washington State University, Pullman, Wash. 99163, USA. Exptl
Cell Res 85 (1974)
The production of new centrioles seems to be under very strict control, since centriole replication ordinarily does not get out of phase with nuclear division and neither too many nor too few centrioles are produced [6]. The use of cell synchronization procedures permits biochemical study of whole populations of cells at specific times in the cell cycle [15-171 and allows the study of organelle structure and function in a precise way. Stubblefield & Brinkley [ll] used synchronized Chinese hamster cells to study the centriole replication cycle. Near the middle of the S phase, centriole replication begins by the formation of ‘daughter’ centrioles (procentrioles) at right angle to the mature or ‘parent’ centriole. The procentrioles continue to grow slowly during S phase and G2 19, 111. During late G2 and prophase there appears to be a burst of growh [18]. The present study was undertaken to de-
termine if procentriole formation depends on DNA or RNA synthesis. Both amethopterin and 5-fluoro-2’-deoxyuridine (FUdR) are known to block DNA synthesis in mammalian cells 119, 20, 211 and actinomycin D is known to block RNA synthesis 115, 221. Thus, procentriole formation can be studied at specific time points after treating synchronized cells with each of these drugs. A similar study of the effects of arabinosyl cytosine, a DNA synthesis inhibitor, was published recently by Rattner & Phillips 11181.Our own study has also appeared in an abstract published earlier 1231. ATERlALS Cell line-characteristics
AND METHODS and maintenance
Cells of the Chinese hamster cloned strain, Don-C, are pseudo-diploid, have very rapid growth, and have a cell generation time of about 12 h. On the average each cell cycle may be subdivided as follows: Gl phase, 2 h: S phase, 7 h; G2 phase, 2.5 h; M, 0.5 h [24$
The cells were grown as monolayers in plastic T75 in McCoy’s 5a medium modified by the addition of lactalbumin hydrolysate (0.8 g/l) and fetal calf serum (100 ml/l). The cells were subcultured daily by washing the cells first with Hanks solution (minus calcium and magnesium), and then trypsin (0.01 %) to remove the ce!ls from the tissue culture flask. Each culture was divided into 3 new cultures in fresh medium, gassed with 10 % CO, in air, and incubated at 37°C. Cultures were started from frozen stocks every 6 to 8 weeks to eliminate the effects of age on cell generation time and ploidy. flasks
Cell synchronization with Colcemid The day before an experiment, cells were subcultured into 600 ml serum bottles (2 T75’s per bottle) in a total of 100 ml of medium. The bottles were rotated at 8 rph in a 37°C incubator. Colcemid (0.06 ,ug/ml) was added for 2 h, approx. 18 h after subculturing, in order to take advantage of the partial synchrony induced by subculturing [16]. Metaphase cells were collected by a 45 set treatment with trypsin (0.01 X, Worthington). They were then centrifuged and resuspended into either fresh medium or medium supplemented with a drus. Anarox. 7 x lo6 metanhase cells were planted in plastic T 75 culture flasks with 20 ml of medium. The cultures were gassed with 10 % COz in air and incubated at 37°C. P;fter 15 min the T75’s were gently shaken and then the suspension of mitotic cells was reseeded into a fresh T75. This increased the purity of the mitotic preparation, since contami-
nating interphase cells rapidly reattach to the flask whereas the mitotic cells do not. Samples of the metaphase cell suspension were taken for each experiment and fixed 3n 50 % acetic acid, and a squash preparation was made to determine the percentage of cells in mitosis. Metaphase ceils comprised 93-96 “/b of the sample for each of the experiments described.
Experimental drugs FIJdR-stock solutions were made in 50 +g/ml concentrations and stored at -20°C. They were thawed when needed and added to cultures- to achieve a final concentration of 1.0 pug/ml. FUdR is an analog of thymidine and blocks DNA synthesis 3y the competitive inhibition of thymidylate synthetase [19-Z]. Amethopterin-Stock solutions of 1QO pug/ml -were stored at - 20°C. A final COW. of 1.O&ml was used in the culture medium. Amethopterin, an antifolic acid agent inhibits DNA synthesis by interfering iyith the methylation of thymidine. The inhibition can be reversed bv the addition of thvmidine 1241. Hyooxanthine (1O.b pg/ml) must also de added -10 &e ~&ures blocked with amethopierin in order to provide the cells with a purine source, since purine synthesis also depends on methylation steps. Actincmycin D-stock solutions of Dactinomycin (0.5 mg/ml) were made in sterile distilled water. Sohitions were made and Iused immediately, since the drug is known to be light-sensitive. Actinomycin D at a concentration of 4.0 pg/ml is knowr, to block 90 Y0of the RNA synthesis in Don-C cells 119. The drug is thought to function by inhibiting transcription by DNA-dependent RNA polymerase [22, 25> 261.
Preparation of cells for electron microscopy Cell samples were collected by trypsinization at 6 h after reversal from the Colcemid biock and fixed in suspension by a 3 % glutaraIdehyde-~illo~ig phosuhate buffer mixture. OH 7.4. at 4°C. The cells were allowed to fix in s&pension for I5 min and then centrifuged at 16000 g for ?5 min to form a pellet convenient for processing. The material was loosened from the walls of the centrifuge tube and fixation was continued for an additional 40 min. The pellet was next washed in Raillonip. vhosohate buffer. 3H 7.4 for 15 min and then post-fiiid in ‘1 % osmium’tktraoxide-Millonig phosphate buffer, p 7.4, at room tempera-, ture, for 30 to 60 min. The cells were then washed in several changes of distilled water and prestained fol 12-16 h with 2 96 aqueous uranyi acetate. The pellet was processed through a graded series of alcohols and embedded in Spurr 1271,or, after the last aicoboi step, processed through propylene oxide and ihen embedded in Epon [28]. Gray-to-silver sections were cut with a Dupont diamond knife on the LK%s Ultrotome PI1 and then placed on slotted copper grids coated with either Formvar or collodion. The sections were stained for 4-7 min with uranyi acetate [29] and then for 1-2 min with lead citrate [3Qj. The stained sections were examined in a Hitachi HS-8 or a Hitach:
138
DeFoor and Stubblefield
Fig. I. Replicating centrioles in a cell from a control culture 6 h after division (S phase). The daughter pr ocentrioles are indicated by arrows. x 60 000. Fig. 2. S phase cell treated with FUdR for 6 h showing procentrioles (nrrows) developing as in the ccantrol cultures. x 60 000. Figs $4. S phase cells treated with amethopterin for 6 h in which daughter procentrioles (arrows) appear to be forming as in the control cultures. x 80 000. HU-11C transmission electron microscope at 50 and 75-100 KV respectively.
operated
Cell and centriole profile counts To insure random samolina of sections to survev for cell and centriole profileco&ts, the sections cut oh the Ultrotome III were collected and surveyed in the following manner: (a) When serial sections were cut, only one section was selected at random, and the total number of cells counted. The same section was surveyed at a higher magnification for centriole profiles; (b) single sections or small serials ( i 4 sections) were cut with a diamond knife on the Ultrotome III: Next a glass knife was used to retrim the block face. Then Exptl Cell Res 85 (1974)
single sections were taken again for survey. This procedure eliminated the possibility of counting the same cells more than one time. Centriole profiles from the selected sections were counted by surveying the cells at higher magnification.
RESULTS Centrioles are usually found in a region near the nucleus in interphase cells (fig. 1). They usually occupy a region relatively free of ribosomes and with elements of the Golgi
NO.
Treatment
No. of cell profiles
containing procentrioles
No. per 1 000
x2
>P>
Control 5-FUdR Amethopterin Actinomycin D
1 120 1 012 1 223 1 051
;
7.1 6.9 3.3
0.012 2.03
0.950-0.900 0.250-0.100
0.95
5.33
0.025-0.030
4 1
apparatus nearby. In the Gl phase of the cell cycle there are two centrioles, usually in a “‘V” shaped orientation. These centrioles begin to replicate in the subsequent S phase so that by the middle of S phase short daughter centrioles are found at right angles to the parent centrioles, as in fig. 1. The daughter centrioles always form at the proximal end of the parent and are separated from the parent by a space of 500-700 A. The daughter centrioles are approx. 1 000 A in length by the middle of the S phase in Don-C cells. The daughter centrioles continue to mature (elongate) in G2 and mitosis and appear to complete maturation in 61 of the next cycle. Calculations made on control cells have shown that centrioles are observed in 6 74 of the cell profiles [31]. Of the centriole profiles observed in mid-S phase, about 10 % appear to be daughter centrioles. FUd
Examination of synchronized cells treated with FUdR for 6 h indicated that daughter centriole formation occurs at a time when the cells would usually be in the middle of the S phase if FUdR were not present and at the same frequency as found in control cells (fig. 2, table I). There were no obvious structural changes in the centrioles of cells treated with FUdR, nor did the orientation of the parent and daughter centrioles vary from those in control cells. These observations in-
dicate that treatment prevent the production
-
with FUd of daughter centrioles,
Amethopterin
Daughter centrioles are also found (figs 3, 4) at a time following reversal equivsiient to the middle of the S phase in cells amethopterin (1.0 lug/ml), but t occur at a lower frequency. Analysis by use of the X-square test showed that the lower frequency of daughter centrioles observed wever, could occur by chance (table I), a lack serial sectioning of cells demons&a of daughter centriole formation in som:: ceils treated with amethopterin (fig. 5). No lice structural changes were found in these cells as a result of amethopterin treatment. Actimmycin D
The frequency of daughter centrioles o served at the middle of the S phase in cel!s treated with actinomycin is extremely low when compared with observations made on control cells. The x-square test shows this to be a significant reduction (table 1). Se sectioning through cells at a time when I would be in the middle of t actinomycin D were not present, shows a lack of daughter centriole formation (fig. 6). This indicates that RNA synt reason necessary for the production of daughter centrioles. In fig. 6 structures are also visible (arrows) which closely resemble foot-
140
DeFoor and Stubblefield
Fig. 5. Serial sections through a pair of centrioles in a cell treated with amethopterin as in figs 3 and 4. In this case no daughter centrioles were found, and the daughter of the previous generation is not full length, suggesting that the drug may have inhibited maturation as well as reproduction in this case [lo]. x 35 000.
lets seen on basal bodies. These have never been seenin control centrioles and may have appeared as a result of the drug treatment. DISCUSSION Whether or not centrioles contain DNA or RNA as part of their structure has been diffiExptl Cell Res 85 (1974)
cult to ascertain. The studies of Randall & Disbrey [30], Smith-Sonnenborn & Plaut [32], Dippell [3], Ackerman [33], Stubblefield & Brinkley [ll], and Seaman [34], to list a few, all contain some evidence in favor of the view that centrioles and basal bodies contain RNA or DNA or both. If nucleic acids were
acid is a structural
component
of centrioles,
Actinomycin 19 might block the formation oi: a nuclear messenger RNA which is necessary for the formation of a structural protein component of centrioles. The effect of aclinomycin which we have observed must now be examined further to decide which of these possibilities is more likely Treatment with 5-Ftid appears 130t to have inhibited procentriole formation. The slight depression in frequency seen after treatment is not statistically owever, serial section anaIysis
did demonstrate
that in some cases procen-
trioles were not found where the petted. Such a result suggests that
thesis inhibitors may not be uniformly effective; but this is not surprising since these drugs are only partial inhibitors and a?!ow some slow DNA replication to QCCX 1353. The authors wish to thank Cheryl Menees arid Nita Rivoire for technical assistance. We are also grateful to Dr B. R. Brinkley for the use of his electron *17icroscope facilities. Tnis work wassupportedby MCI Training Grant CA-5047from USPHS.
EFERENCES
Fig. 6. Serial sections through a centriole in a ceil treated with actinomycin D for 6 h. No daughter procentrioie is present. Structures resembling basal body rootlets are visible (arrolus); these may have been produced in response to the drug treatment. x 35 000.
1. Wilson, E B, The cell in developmeni,p. 165. Macmillan,New York (1928). 2. DeHarven,E, The nucleus(ed A J Dalton & F Hagenau)p. 197. AcademicPress,Xew York (1968).
ocnatl acadsciUS 61(1968)461 4. Dirkson.E R. J ceilbiol51 (1971)286. 5. Dirkson; E g & Cracker, T‘ T’, J’microsc 5 (3965) 629.
structural components of centrioles, then .one might expect inhibition of DNA or RNA synthesis to block procentriole formation. It is still possible to imagine that a block in nucleic acid synthesis might not have an immediate effect, but might block replication of a defective procentriole in a later generalion. On the other hand, a positive effect of an ibilor does not prove that the nucleic
6. F&on, C, Origin andcontinuityof cellorganelles (ed J Reinert & H Ursprung)p. 170.Springer, Berlin (1971). 7. Hoage, T R & Kessel, R G, J ultrastruct res 24 (1968) 6. 8. Kalnins, V L & Porter, M R, Z ZeUforsch 100 (1969) 1. 9. Robdins, E, Jentzsch, 6 & Micaii, A, J cell biol 36 (1968) 329. 10. Stubblefield, E, The proliferation and spread of neoplastic cells, p. 175. William5 81 Wilkins, Baltimore, Md (1967). 11. Stubblefield, E & Brinkley, B R, Formation and fate of cell organelles (ed K % Warren) p, 175. Academic Press, New York (1967).
142
DeFoor and Stubblefield
12. Sorokin, S P, J cell sci 3 (1968) 207. 13. Steinman, R M, Am j anat 122 (1968) 19. 14. Mazia, D, Harris, P J & Bibring, T, J biophys biochem cytol 7 (1960) 1. 15. Murphree, S, Stubblefield, E & Moore, E C, Exptl cell res 58 (1969) 118. 16. Stubblefield, E, Klevecz, R & Deaven, L, J cell physiol 69 (1967) 345. 17. Stubblefield, E & Gay, M, Chromosoma 31 (1970) 79. 18. Rattner, J B & Phillips, S G, J cell biol 57 (1973) 359. 19. Hartman, K U & Heidelberger, C J, Biol them 236 (1962) 3006. 20. Ockey, C H, Hsu, T C & Richardson, L C, J natl cancer inst 40 (1968) 465. 21. Rueckert, R R & Mueller, G C, Cancer res 20 (1960) 1584. 22. Reich, E, Science 143 (1964) 685. 23. Stubblefield, E & DeFoor, P H, J cell biol 55 (1972) 254a (abst.) 24. Stubblefield, E, Methods in cell physiol 3 (1968) 25.
Exptl
Cell Res 85 (1974)
25. Arrighi, F E & Hsu, T C, Exptl cell res 39 (1965) 305. 26. Harbers, E & Muller, W, Biochem biophys res comm 7 (1962) 107. 27. Spurr, A R, J ultrastruct res 26 (1969) 31. 28. Luft, J H, J biophys biochem cytol 9 (1961) 409. 29. Watson, M L, J biophys biochem cytol 4 (1958) 475. 30. Randall, J T & Disbrey, C, Proc roy sot B 162 (1965) 473. 31. Mullins, R & Wette, R, J cell biol 30 (1966) 652. 32. Smith-Sonneborn. J & Plaut. W, J cell sci 2 (1967) 225. 33. Ackerman, G A, J biophys biochem cytol 11 (1961) 717. 34. Seaman, G R, Exptl cell res 21 (1960) 292. 35. Meyn, R E, Hewitt, R R & Humphrey, R M, Exptl cell res 82 (1973) 137.
Received September 27, 1973
Experimental
EFFECTS AND
Cell Research 85 (1974) 136-142
OF ACTINOMYCIN
5-FLURO-2’-DEOXYURIDINE IN
CHINESE
HAMSTER P. H. DeFOORl
The University
D, AMETHOPTERIN, ON PROCENTRIOLE
FIBROBLASTS
IN
FORMATION CULTURE
and E. STUBBLEFIELD
of Texas M. D. Anderson Hospital and Tumor Institute, and the Graduate of Biomedical Sciences, Houston, Tex. 77025, USA
School
SUMMARY Synchronized Chinese hamster fibroblasts (Don-C) were cultured for 6 h in the presence of amethopterin, 5-fluorodeoxyuridine (FUdR), or actinomycin D and were then examined by electron microscopy of thin sections for the presence or absence of daughter procentrioles. Control cells were at mid S phase and contained obvious procentrioles in about 1 profile out of 140. Treatment with FUdR or amethopterin did not reduce this frequency significantly. However, serial section analysis did demonstrate a lack of procentrioles in some cells treated with amethopterin. Actinomycin was quite effective in preventing daughter procentriole formation.
Centrioles were first observed in 1886 by Van Beneden & Boveri [l]. However, studies of the fine structure and mode of replication of these minute organelles were hindered until the advent of electron microscopy in the 1950s. Since then, many studies on centriole structure and function [2-111 have led to speculations that centrioles might be selfreplicating structures containing centriolespecific DNA. However, it has not yet been possible to discern this with certainty, and investigations of the factors involved in centriole replication are needed to further our understanding in this regard. In dividing cells, the diplosomal centrioles replicate in each cell cycle and are separated at mitosis in such a way that each newly divided cell has two centrioles [l, 10, 11, 141. 1 Current address: Department of Veterinary Microbiology, Washington State University, Pullman, Wash. 99163, USA. Exptl
Cell Res 85 (1974)
The production of new centrioles seems to be under very strict control, since centriole replication ordinarily does not get out of phase with nuclear division and neither too many nor too few centrioles are produced [6]. The use of cell synchronization procedures permits biochemical study of whole populations of cells at specific times in the cell cycle [15-171 and allows the study of organelle structure and function in a precise way. Stubblefield & Brinkley [ll] used synchronized Chinese hamster cells to study the centriole replication cycle. Near the middle of the S phase, centriole replication begins by the formation of ‘daughter’ centrioles (procentrioles) at right angle to the mature or ‘parent’ centriole. The procentrioles continue to grow slowly during S phase and G2 19, 111. During late G2 and prophase there appears to be a burst of growh [18]. The present study was undertaken to de-
termine if procentriole formation depends on DNA or RNA synthesis. Both amethopterin and 5-fluoro-2’-deoxyuridine (FUdR) are known to block DNA synthesis in mammalian cells 119, 20, 211 and actinomycin D is known to block RNA synthesis 115, 221. Thus, procentriole formation can be studied at specific time points after treating synchronized cells with each of these drugs. A similar study of the effects of arabinosyl cytosine, a DNA synthesis inhibitor, was published recently by Rattner & Phillips 11181.Our own study has also appeared in an abstract published earlier 1231. ATERlALS Cell line-characteristics
AND METHODS and maintenance
Cells of the Chinese hamster cloned strain, Don-C, are pseudo-diploid, have very rapid growth, and have a cell generation time of about 12 h. On the average each cell cycle may be subdivided as follows: Gl phase, 2 h: S phase, 7 h; G2 phase, 2.5 h; M, 0.5 h [24$
The cells were grown as monolayers in plastic T75 in McCoy’s 5a medium modified by the addition of lactalbumin hydrolysate (0.8 g/l) and fetal calf serum (100 ml/l). The cells were subcultured daily by washing the cells first with Hanks solution (minus calcium and magnesium), and then trypsin (0.01 %) to remove the ce!ls from the tissue culture flask. Each culture was divided into 3 new cultures in fresh medium, gassed with 10 % CO, in air, and incubated at 37°C. Cultures were started from frozen stocks every 6 to 8 weeks to eliminate the effects of age on cell generation time and ploidy. flasks
Cell synchronization with Colcemid The day before an experiment, cells were subcultured into 600 ml serum bottles (2 T75’s per bottle) in a total of 100 ml of medium. The bottles were rotated at 8 rph in a 37°C incubator. Colcemid (0.06 ,ug/ml) was added for 2 h, approx. 18 h after subculturing, in order to take advantage of the partial synchrony induced by subculturing [16]. Metaphase cells were collected by a 45 set treatment with trypsin (0.01 X, Worthington). They were then centrifuged and resuspended into either fresh medium or medium supplemented with a drus. Anarox. 7 x lo6 metanhase cells were planted in plastic T 75 culture flasks with 20 ml of medium. The cultures were gassed with 10 % COz in air and incubated at 37°C. P;fter 15 min the T75’s were gently shaken and then the suspension of mitotic cells was reseeded into a fresh T75. This increased the purity of the mitotic preparation, since contami-
nating interphase cells rapidly reattach to the flask whereas the mitotic cells do not. Samples of the metaphase cell suspension were taken for each experiment and fixed 3n 50 % acetic acid, and a squash preparation was made to determine the percentage of cells in mitosis. Metaphase ceils comprised 93-96 “/b of the sample for each of the experiments described.
Experimental drugs FIJdR-stock solutions were made in 50 +g/ml concentrations and stored at -20°C. They were thawed when needed and added to cultures- to achieve a final concentration of 1.0 pug/ml. FUdR is an analog of thymidine and blocks DNA synthesis 3y the competitive inhibition of thymidylate synthetase [19-Z]. Amethopterin-Stock solutions of 1QO pug/ml -were stored at - 20°C. A final COW. of 1.O&ml was used in the culture medium. Amethopterin, an antifolic acid agent inhibits DNA synthesis by interfering iyith the methylation of thymidine. The inhibition can be reversed bv the addition of thvmidine 1241. Hyooxanthine (1O.b pg/ml) must also de added -10 &e ~&ures blocked with amethopierin in order to provide the cells with a purine source, since purine synthesis also depends on methylation steps. Actincmycin D-stock solutions of Dactinomycin (0.5 mg/ml) were made in sterile distilled water. Sohitions were made and Iused immediately, since the drug is known to be light-sensitive. Actinomycin D at a concentration of 4.0 pg/ml is knowr, to block 90 Y0of the RNA synthesis in Don-C cells 119. The drug is thought to function by inhibiting transcription by DNA-dependent RNA polymerase [22, 25> 261.
Preparation of cells for electron microscopy Cell samples were collected by trypsinization at 6 h after reversal from the Colcemid biock and fixed in suspension by a 3 % glutaraIdehyde-~illo~ig phosuhate buffer mixture. OH 7.4. at 4°C. The cells were allowed to fix in s&pension for I5 min and then centrifuged at 16000 g for ?5 min to form a pellet convenient for processing. The material was loosened from the walls of the centrifuge tube and fixation was continued for an additional 40 min. The pellet was next washed in Raillonip. vhosohate buffer. 3H 7.4 for 15 min and then post-fiiid in ‘1 % osmium’tktraoxide-Millonig phosphate buffer, p 7.4, at room tempera-, ture, for 30 to 60 min. The cells were then washed in several changes of distilled water and prestained fol 12-16 h with 2 96 aqueous uranyi acetate. The pellet was processed through a graded series of alcohols and embedded in Spurr 1271,or, after the last aicoboi step, processed through propylene oxide and ihen embedded in Epon [28]. Gray-to-silver sections were cut with a Dupont diamond knife on the LK%s Ultrotome PI1 and then placed on slotted copper grids coated with either Formvar or collodion. The sections were stained for 4-7 min with uranyi acetate [29] and then for 1-2 min with lead citrate [3Qj. The stained sections were examined in a Hitachi HS-8 or a Hitach:
138
DeFoor and Stubblefield
Fig. I. Replicating centrioles in a cell from a control culture 6 h after division (S phase). The daughter pr ocentrioles are indicated by arrows. x 60 000. Fig. 2. S phase cell treated with FUdR for 6 h showing procentrioles (nrrows) developing as in the ccantrol cultures. x 60 000. Figs $4. S phase cells treated with amethopterin for 6 h in which daughter procentrioles (arrows) appear to be forming as in the control cultures. x 80 000. HU-11C transmission electron microscope at 50 and 75-100 KV respectively.
operated
Cell and centriole profile counts To insure random samolina of sections to survev for cell and centriole profileco&ts, the sections cut oh the Ultrotome III were collected and surveyed in the following manner: (a) When serial sections were cut, only one section was selected at random, and the total number of cells counted. The same section was surveyed at a higher magnification for centriole profiles; (b) single sections or small serials ( i 4 sections) were cut with a diamond knife on the Ultrotome III: Next a glass knife was used to retrim the block face. Then Exptl Cell Res 85 (1974)
single sections were taken again for survey. This procedure eliminated the possibility of counting the same cells more than one time. Centriole profiles from the selected sections were counted by surveying the cells at higher magnification.
RESULTS Centrioles are usually found in a region near the nucleus in interphase cells (fig. 1). They usually occupy a region relatively free of ribosomes and with elements of the Golgi
NO.
Treatment
No. of cell profiles
containing procentrioles
No. per 1 000
x2
>P>
Control 5-FUdR Amethopterin Actinomycin D
1 120 1 012 1 223 1 051
;
7.1 6.9 3.3
0.012 2.03
0.950-0.900 0.250-0.100
0.95
5.33
0.025-0.030
4 1
apparatus nearby. In the Gl phase of the cell cycle there are two centrioles, usually in a “‘V” shaped orientation. These centrioles begin to replicate in the subsequent S phase so that by the middle of S phase short daughter centrioles are found at right angles to the parent centrioles, as in fig. 1. The daughter centrioles always form at the proximal end of the parent and are separated from the parent by a space of 500-700 A. The daughter centrioles are approx. 1 000 A in length by the middle of the S phase in Don-C cells. The daughter centrioles continue to mature (elongate) in G2 and mitosis and appear to complete maturation in 61 of the next cycle. Calculations made on control cells have shown that centrioles are observed in 6 74 of the cell profiles [31]. Of the centriole profiles observed in mid-S phase, about 10 % appear to be daughter centrioles. FUd
Examination of synchronized cells treated with FUdR for 6 h indicated that daughter centriole formation occurs at a time when the cells would usually be in the middle of the S phase if FUdR were not present and at the same frequency as found in control cells (fig. 2, table I). There were no obvious structural changes in the centrioles of cells treated with FUdR, nor did the orientation of the parent and daughter centrioles vary from those in control cells. These observations in-
dicate that treatment prevent the production
-
with FUd of daughter centrioles,
Amethopterin
Daughter centrioles are also found (figs 3, 4) at a time following reversal equivsiient to the middle of the S phase in cells amethopterin (1.0 lug/ml), but t occur at a lower frequency. Analysis by use of the X-square test showed that the lower frequency of daughter centrioles observed wever, could occur by chance (table I), a lack serial sectioning of cells demons&a of daughter centriole formation in som:: ceils treated with amethopterin (fig. 5). No lice structural changes were found in these cells as a result of amethopterin treatment. Actimmycin D
The frequency of daughter centrioles o served at the middle of the S phase in cel!s treated with actinomycin is extremely low when compared with observations made on control cells. The x-square test shows this to be a significant reduction (table 1). Se sectioning through cells at a time when I would be in the middle of t actinomycin D were not present, shows a lack of daughter centriole formation (fig. 6). This indicates that RNA synt reason necessary for the production of daughter centrioles. In fig. 6 structures are also visible (arrows) which closely resemble foot-
140
DeFoor and Stubblefield
Fig. 5. Serial sections through a pair of centrioles in a cell treated with amethopterin as in figs 3 and 4. In this case no daughter centrioles were found, and the daughter of the previous generation is not full length, suggesting that the drug may have inhibited maturation as well as reproduction in this case [lo]. x 35 000.
lets seen on basal bodies. These have never been seenin control centrioles and may have appeared as a result of the drug treatment. DISCUSSION Whether or not centrioles contain DNA or RNA as part of their structure has been diffiExptl Cell Res 85 (1974)
cult to ascertain. The studies of Randall & Disbrey [30], Smith-Sonnenborn & Plaut [32], Dippell [3], Ackerman [33], Stubblefield & Brinkley [ll], and Seaman [34], to list a few, all contain some evidence in favor of the view that centrioles and basal bodies contain RNA or DNA or both. If nucleic acids were
acid is a structural
component
of centrioles,
Actinomycin 19 might block the formation oi: a nuclear messenger RNA which is necessary for the formation of a structural protein component of centrioles. The effect of aclinomycin which we have observed must now be examined further to decide which of these possibilities is more likely Treatment with 5-Ftid appears 130t to have inhibited procentriole formation. The slight depression in frequency seen after treatment is not statistically owever, serial section anaIysis
did demonstrate
that in some cases procen-
trioles were not found where the petted. Such a result suggests that
thesis inhibitors may not be uniformly effective; but this is not surprising since these drugs are only partial inhibitors and a?!ow some slow DNA replication to QCCX 1353. The authors wish to thank Cheryl Menees arid Nita Rivoire for technical assistance. We are also grateful to Dr B. R. Brinkley for the use of his electron *17icroscope facilities. Tnis work wassupportedby MCI Training Grant CA-5047from USPHS.
EFERENCES
Fig. 6. Serial sections through a centriole in a ceil treated with actinomycin D for 6 h. No daughter procentrioie is present. Structures resembling basal body rootlets are visible (arrolus); these may have been produced in response to the drug treatment. x 35 000.
1. Wilson, E B, The cell in developmeni,p. 165. Macmillan,New York (1928). 2. DeHarven,E, The nucleus(ed A J Dalton & F Hagenau)p. 197. AcademicPress,Xew York (1968).
ocnatl acadsciUS 61(1968)461 4. Dirkson.E R. J ceilbiol51 (1971)286. 5. Dirkson; E g & Cracker, T‘ T’, J’microsc 5 (3965) 629.
structural components of centrioles, then .one might expect inhibition of DNA or RNA synthesis to block procentriole formation. It is still possible to imagine that a block in nucleic acid synthesis might not have an immediate effect, but might block replication of a defective procentriole in a later generalion. On the other hand, a positive effect of an ibilor does not prove that the nucleic
6. F&on, C, Origin andcontinuityof cellorganelles (ed J Reinert & H Ursprung)p. 170.Springer, Berlin (1971). 7. Hoage, T R & Kessel, R G, J ultrastruct res 24 (1968) 6. 8. Kalnins, V L & Porter, M R, Z ZeUforsch 100 (1969) 1. 9. Robdins, E, Jentzsch, 6 & Micaii, A, J cell biol 36 (1968) 329. 10. Stubblefield, E, The proliferation and spread of neoplastic cells, p. 175. William5 81 Wilkins, Baltimore, Md (1967). 11. Stubblefield, E & Brinkley, B R, Formation and fate of cell organelles (ed K % Warren) p, 175. Academic Press, New York (1967).
142
DeFoor and Stubblefield
12. Sorokin, S P, J cell sci 3 (1968) 207. 13. Steinman, R M, Am j anat 122 (1968) 19. 14. Mazia, D, Harris, P J & Bibring, T, J biophys biochem cytol 7 (1960) 1. 15. Murphree, S, Stubblefield, E & Moore, E C, Exptl cell res 58 (1969) 118. 16. Stubblefield, E, Klevecz, R & Deaven, L, J cell physiol 69 (1967) 345. 17. Stubblefield, E & Gay, M, Chromosoma 31 (1970) 79. 18. Rattner, J B & Phillips, S G, J cell biol 57 (1973) 359. 19. Hartman, K U & Heidelberger, C J, Biol them 236 (1962) 3006. 20. Ockey, C H, Hsu, T C & Richardson, L C, J natl cancer inst 40 (1968) 465. 21. Rueckert, R R & Mueller, G C, Cancer res 20 (1960) 1584. 22. Reich, E, Science 143 (1964) 685. 23. Stubblefield, E & DeFoor, P H, J cell biol 55 (1972) 254a (abst.) 24. Stubblefield, E, Methods in cell physiol 3 (1968) 25.
Exptl
Cell Res 85 (1974)
25. Arrighi, F E & Hsu, T C, Exptl cell res 39 (1965) 305. 26. Harbers, E & Muller, W, Biochem biophys res comm 7 (1962) 107. 27. Spurr, A R, J ultrastruct res 26 (1969) 31. 28. Luft, J H, J biophys biochem cytol 9 (1961) 409. 29. Watson, M L, J biophys biochem cytol 4 (1958) 475. 30. Randall, J T & Disbrey, C, Proc roy sot B 162 (1965) 473. 31. Mullins, R & Wette, R, J cell biol 30 (1966) 652. 32. Smith-Sonneborn. J & Plaut. W, J cell sci 2 (1967) 225. 33. Ackerman, G A, J biophys biochem cytol 11 (1961) 717. 34. Seaman, G R, Exptl cell res 21 (1960) 292. 35. Meyn, R E, Hewitt, R R & Humphrey, R M, Exptl cell res 82 (1973) 137.
Received September 27, 1973