CELL PROLIFERATION IN COMPLEX TISSUES: THE CONTROL OF THE MITOTIC CYCLE OF CELL POPULATIONS IN THE CULTURED ROOT MERISTEM OF SUNFLOWER (HELIANTHUS)

Experiments were performed with cultured primary root tips of sunflower (Helianthus annuus var. Russian Mammoth) to determine: (1) if progression in the mitotic cycle of meristematic cells was nutritionally controllable by carbohydrate starvation and replenishment; (2) where in the mitotic cycle control was effected; and (3) whether nutritional deprivation could be used to detect phenotypically different subpopulations in a complex tissue. Meristematic cells were rendered stationary by carbohydrate starvation, as indicated by the absence of cell division; this condition was reversed by carbohydrate provision. After 72 or 96 hr of starvation most cells stopped in G1 (80–90%) and G2 (10–20%), and a very few (“leaky” cells) continued to enter S. “Leaky” cells represent a small population with an S period of approximately 4.1 hr that either lack a principal control point in G1 or have an unusual metabolism whereby the control point requirements are met and have a carbohydrate dependence for mitosis. Though phenotypically different, no specific functions can be attributed to “leaky” cells at this time.     

Timing and Regulation of Nuclear and Cortical Events in the Cell Cycle of Tetrahymena pyriformis*

SYNOPSIS. Using continuous flow cultures based on the chemostat principle, we varied the cell generation times of the ciliate Tetrahymena pyriformis strain GL, from 4.9 to 22.2 hr and studied various parameters of the cell cycle at 28 C. These included: the duration of the periods required for oral morphogenesis, macronuclear division, cell division, G₁ S, and G₂. The size of individual cells was also measured. Independent of the growth rate, the period of oral morphogenesis occurred during the last 90 min of the cell cycle. In all cases macronuclear and cell divisions took place during the last part of these 90 min, and the final macronuclear separation occurred just before final cell separation. The S-period increased slightly, while the G₁ and G₂ both increased in roughly the same relative proportion to the increasing generation times. Slowly growing cells (generation time 20.5 hr) were shorter but broader and somewhat larger in volume than quickly growing cells (generation time 4.9 hr).     

Preuves Cinétiques de l'Existence d'une Période de Compétence pour la Transformation Microstome-Macrostome dans le Cycle Cellulaire de Tetrahymena paravorax

RÉSUMÉ. Des expérimentations portant sur des cellules isolées ont montré que 77% des microstomes de Tetrahymena paravorax, prélevés au hasard dans des cultures en phase logarithmique de croissance, se transforment directement en macrostomes en présence de “stomatine.” Ces macrostomes apparaissent à des moments variés entre 1,5 et 9 h après l'addition de la stomatine (“point 50% de transformations” vers 2,5 h). La compétence pour le remplacement oral est en relation avec la position dans le cycle cellulaire. Les pourcentages de transformation les plus élevés sont observés avec les populations testées pendant la première moitié de la période moyenne d'interfission. La formation des macrostomes est d'autant plus rapide que l'ǎge initial des microstomes est plus proche du point médian du cycle cellulaire (“point de compétence”). Dans la seconde moitié de ce cycle, le temps moyen de transformation reste à peu près constant, mais le pourcentage de divisions augmente: le “point de transition” (50% de divisions) se trouve au début d'une phase terminale représentant 19–20% de la durée totale du cycle. La transformation des produits de bipartition antérieur et postérieur est nettement asynchrone: dans la majorité des paires cellulaires, l'opisthe se transforme avant le proter. Les cellules-soeurs se divisent aussi de manière asynchrone: le temps de génération du proter est plus long que celui de l'opisthe. Le problème de l'acquisition de la compétence pour le changement de phénotype est discuté en envisageant les corrélations éventuelles avec certains processus majeurs du cycle cellulaire. SYNOPSIS. Seventy-seven percent of the microstomes of Tetrahymena paravorax taken from random samples of log-phase cultures transform directly into macrostomes in the presence of “stomatin.” These macrostomes appear between 1.5 and 9 h after addition of stomatin (“50% transformation point,”～ 2.5 h). Competence for oral replacement is related to the position in the cell cycle. The highest percentages of transformation are observed in populations tested during the first half of the mean interfission period. Formation of macrostomes is more rapid when the initial age of the microstomes is nearer to the midpoint of the cell cycle (“competence point”). In the 2nd half of this cycle, the mean transformation time remains approximately constant, but the percentage of cells undergoing division is increasing. The “transition point” (50% divided cells) is found at the beginning of a terminal phase which accounts for 19–20% of the cell cycle. Transformation of anterior and posterior fission products is fairly asynchronous; in the majority of individual pairs, the opisthe is transformed before the proter. The daughter cells also divide asynchronously, the generation time of the proter being longer than that of the opisthe. The problem of acquisition of competence for phenotypic change is discussed in light of possible correlations with certain major processes of the cell cycle.     

GROWTH AND CELL CYCLE OF TWO CLOSELY RELATED RED TIDE-FORMING DINOFLAGELLATES: GYMNODINIUM NAGASAKIENSE AND G. CF. NAGASAKIENSE1

Small-sized vegetative cells were found to co-occur with normal-sized cells in populations of the European bloom-forming dinoflagellate Gymnodinium cf. nagasakiense Takayama et Adachi, currently known as Gyrodinium aureolum Hulburt, but not in populations of the closely related Japanese species Gymnodiniumn agasakiense. We examined how cell size differentiation may influence growth and cell cycle progression under a 12:12-h light: dark cycle in the European taxon, as compared to the Japanese one. Cell number and volume and chlorophyll red fluorescence in both species varied widely during the photocycle. These variations generally appeared to be related lo the division period, which occurred at night, as indicated by the variations of the fraction of binucleated cells (mitotic index) as well as the distribution of cellular DNA content. “Small” cells of G. cf. nagasakiense divided mainly during the first part of the dark period, although a second minor peak of dividing cells could occur shortly before light onset. In contrast, “large” cells displayed a sharp division peak that occurred 9 h after the beginning of the dark period. The lower degree of synchrony of “small” cells could be a consequence of their faster growth. Alternatively, these data may suggest that cell division is lightly controlled by an endogenous clock in “large” cells and much more loosely controlled in “small” cells. Cells of the Japanese species, which were morphologically similar to “large” cells of the European taxon, displayed an intermediate growth pattern between the two cell types of G. cf. nagasakiense, with a division period that extended to most of the dark period.     

A mammalian somatic "cell cycle" mutant defective in G1

Variants or “mutants” temperature-sensitive (ts) for growth have been isolated by selection from a near-diploid mouse cell line. Thus far. 10 ts mutants which grow normally at 33° C, but not at 39° C, have been isolated. These ts mutants were then studied to determine if any manifested their defect at a unique point or stage in the cell cycle. This type of ts mutant is termed a “cell cycle” mutant. The first screen involves observing individual cells of an asynchronous culture for residual division after a shift from 33° C (permissive temperature) to 39° (nonpermissive temperature). A cell cycle mutant should show some fraction of the cells dividing only once at a normal rate after the shift. The ts variant B54 met this first criterion for a cell cycle mutant (i.e., 50% residual division) and was further analyzed. The second screening technique monitors (1) the rate of entry into S, (2) the length of G₂, and (3) the rate and duration of cells entering mitosis after a shift of an asynchronous culture to 39°. This experiment with B54 revealed that cells in G₁ at the time of the shift to 39° failed to enter S while cells already into S completed the cycle at 39°. These results suggest that B54 is defective in a G₁ function which is required for entry into S, but which is no longer needed once cells have entered S. Other results are presented which also support this hypothesis. In addition the ts function of B54 is apparently required for recovery from a “high density” G₁ arrest.     

Inhibition of erythropoiesis in the spleens of irradiated mice injected with allogeneic lymphocytes. Reactivity of lymphocytes aganist non-H-2 transplantation antigens

Cell interaction requirements for generation of primary IgM, IgG and IgA responses to heterologous erythrocytes in mouse spleen cell cultures have been investigated. Interactions among antigen, macrophages, “helper” thymus-derived cells and precursors of antibody-producing cells are required and are facilitated by incubation of cultures on a rocking platform. Macrophages are required in the cultures for 48 hr for generation of optimal IgM, IgG and IgA responses. Intact erythrocyte antigen is necessary for 48 hr for development of optimal IgM responses, and for 72 hr for optimal IgG and IgA responses. Precursors of IgM antibody-producing cells appear to be “activated” by 48 hr incubation; precursors of IgG and IgA antibody-producing cells appear to be “activated” by 72 hr. These “activated” precursor cells can subsequently undergo final cycles of cell division and differentiate into mature antibody producing cells when incubated stationary in the presence of very few macrophages and in the absence of intact erythrocyte antigen.     

Mechanisms of antibody formation: I. Early in vivo proliferation of mouse spleen T cells as an initial step in antibody formation

Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10⁶?2 × 10⁸ sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement.Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr.Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function.In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10⁸ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.     

DIVISION SYNCHRONY IN PRIMARY CULTURES OF AN ENDOZOIC DINOFLAGELLATE1

Division cysts of zooxanthellae harbored by Anthopleura elegantissima were isolated from random host-associated populations by their preferential attachment to plastic surfaces. By selecting cysts at similar stages in the cell cycle, synchronous division, excystation, and daughter cell reencystment were obtained in a medium enriched with amino acids. The interval of DNA synthesis in a single growth cycle was determined by pulse-labeling with radioactive thymidine. Analysis of the sequence of morphologic changes within such cycles suggested that the initial selection procedure isolates G₂ cells. The transit time from first generation G₂ division cysts to subsequent reentry into G₂ was about 70 hr.     

Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium

Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA⁺ or cnrB⁺) of the donor 9–24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells. In unstable transformation, injected macronucleoplasm is distributed only to one of the daughter cells at every division until about the fifth to seventh fission after injection and then begins to assort equally to daughter cells. The cell cycle stage at injection may influence the mode of transformation. Interspecific microinjection of macronucleoplasm from P. multimicronucleatum and P. tetraurelia to P. caudatum. resulted in the expression of foreign genes in P. caudatum. In one case, injection of macronucleoplasm of P. tetraurelia produced a stable transformant indicating replication of foreign macronucleoplasm in P. caudatum. This work reveals the mode of transformation by injected macronucleoplasm and shows the possibility of transformation among Paramecium species, which is significant in the study of the conservation of gene products and the mechanism of gene expression in different species. © 1992 Wiley-Liss, Inc.     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

DIATOM MINERALIZATION OF SILICIC ACID. II. REGULATION OF Si (OH)4 TRANSPORT RATES DURING THE CELL CYCLE OF NAVICULA PELLICULOSA1

Synchronized populations of Navicula pelliculosa (Bréb.) Hilse show a 10-fold increase in Si(OH)₄ transport rate during traverse through the cell division cycle. The transport activity pattern is similar to a “peak enzyme.” Kinetic analysis showed there was a significant change in K_s values, indicating increased “affinity” for Si(OH)₄ as cells neared maximal uptake rates. However, the dramatic changes in transport rate at various cell cycle stages were also reflected by alterations in the V_max, values of the transport process, suggesting a change in the number of functional transport “sites” in the plasma membrane. Cells in the wall forming stage, arrested from further development by Si(OH)₄ deprivation, maintained high transport rates for as long as 7 h. The rates decreased rapidly if protein synthesis were blocked or if Si(OH)₄ was added, the latter allowing the cells to traverse the rest of the cycle. The half-life of the transport activity ranged from 1.0 to 2.2 h when protein synthesis was inhibited at various cell cycle stages and during the natural decline of activity late in the cycle. The transport system appears to be metabolically unstable as is typical for a “peak protein.” The rise in transport rate through the cell cycle did not depend on the presence of Si(OH)₄ in the medium; therefore, the transport system does not appear to be induced by its substrate. The rise in transport is also observed in L:D synchronized cells developing in the presence of Si(OH)₄; neither does the transport system appear to be derepressed. The transport rate was strongly cell cycle-stage dependent; the data appeared to fit the “dependent pathway” model proposed by Hart-well to explain oscillations in enzyme synthesis during the cell cycle.     

Analysis of mating type determination by transplantation of O macronuclear karyoplasm in Paramecium tetraurelia

The odd (O) or even (E) mating type in Paramecium tetraurelia is determined during the first cell cycle after new macronuclear development. The present paper demonstrates that mating type E is irreversibly determined at the end of the first cell cycle. Direct evidence comes from transplanting O macronuclear karyoplasm containing O-determining factor into E autogamous cells during a new postzygotic macronuclear development. Transplantation of O macronuclear karyoplasm into E autogamous cells at 7–8 hr after the origin of the macronucleus from a product of the synkaryon produces nearly 100% O mating type among the exautogamous cell lines but almost none 10–11 hr after the origin of the macronucleus (around the end of the first cell cycle). The macronuclear anlagen at the stage at which mating type E seems to be fixed contains about 20 times as much DNA as the vegetative G1 micronucleus. The O-determining factor shifting E cells toward O mating type by transplanting O macronuclear karyoplasm is also produced by the newly developed macronucleus in an effective concentration at 10–11 hr after the sensitive period and produced at full levels by the third cell cycle. The level of O factor in the macronucleus then gradually declines with subsequent repeated rounds of DNA synthesis and is finally lost by the eighth cell cycle.     

Critical Phases of Oral Primordium Development in Tetrahymena pyriformis GL-C: An Analysis Employing Low Temperature Treatment

SYNOPSIS. The effects of temperatures of 12–18 C on cell division and oral primordium development were investigated in cultures of synchronized Tetrahymena pyriformis GL-C. If exposures to 12 or 15 C were initiated prior to a “transition point,” long delays of cell division were generated. After this transition point, cell division could no longer be substantially delayed by exposure to low temperature. The time of the transition point was somewhat earlier with 15 C than with 12 C treatments. At temperatures higher than 15 C long delays of cell division were not generated regardless of time of treatment. The effects of low temperature on oral morphogenesis were strongly dependent on the stage which was affected. (i) The further development of cells initially in the “anarchic field” stage (stage 1) was immediately blocked at both 12 and 15 C. (ii) Cells initially in the stages of incipient membranelle differentiation (stages 2 and 3) continued to develop at both 12 and 15 C, and formed oral primordia in which all 3 membranelles were clearly differentiated (stage 4). The subsequent progress of these stage 4 primordia depended on the temperature: at 12 C virtually all were resorbed (and cell division was blocked); at 15 C only about 1/3 were resorbed, while the remaining 2/3 completed their development (with the concomitant completion of cell division). (iii) Cells initially in intermediate stages of membranelle differentiation (early stage 4) developed to some extent at 12 C, and then underwent resorpton of oral primordia and blockage of cell division; at 15 C such cells completed their development and division normally. (iv) Cells in which the membranelles and undulating membrane were complete or nearly so (stage 5 and very late stage 4) at the time of the beginning of the cold treatment subsequently finished their development and went thru cell division, even at temperatures as low as 5 C. These results indicate that in addition to a “stabilization point” which occurs shortly before the completion of membranelle development, there is an earlier change in the primordium at the time of the onset of membranelle development, which renders development much less sensitive to direct interference by low temperature.     

DURATION OF CELL CYCLES IN CULTURED ROOTS OF CONVOLVULUS

The durations of the cell cycle in physiologically different regions of the meristem of cultured roots of Convolvulus arvensis were determined by the metaphase-accumulation technique involving colchicine. The cell cycle in the root cap increases from 13 hr in the actively dividing initials of the first tier to 155 hr in the slowly dividing initials of tiers 2–4 to an indeterminate value for derivatives of the initials in the root cap columella. The cycle times for the cells of the central cylinder and cortex are 21 and 27 hr, respectively. The cells of the quiescent center have a cycle of an estimated 420 hr. The duration of the cell cycle in these different regions is discussed in relation to the increased duration of G₁ in slowly or non-dividing cells. The possible regulation of cell division by the synthesis of a cell-division factor in the quiescent center is also discussed.     

The cell cycle thermal-inactivation sensitive stage of Schizosaccharomyces pombe is independent of 2-phenylethanol-induced changes in S phase location

Synchronous cultures of the fission yeast Schizosaccharomyces pombe 972 h⁻¹ are most sensitive to killing by 15 min, 49 °C pulses during a stage stretching from nuclear division through short G1 and S phases to a point early in G2. In this work the cell cycle position of the S phase has been altered by growing the cells in the presence of 2-phenylethanol. The heat sensitivity of these cells was greater at all stages of the cell cycle compared with the cells grown without 2-phenylethanol. However, the position of the most heat sensitive stage in the cell cycle was unaltered. This heat sensitive stage did not include S phase in the cells grown with 2-phenylethanol.     

Cyclic AMP-induced differentiated neuroblastoma cells: changes in total nucleic acid and protein contents

The total DNA contents of neuroblastoma cells “differentiated” by dibutyryl cyclic AMP, prostaglandin E₁ and 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone treatment was about 50 percent that of control cells, indicating that cells were accumulated in the G₁-phase of the cell cycle. Sodium butyrate-treated cells were also accumulated in the G₁-phase; however, the expression of “differentiated” phenotype did not occur indicating that inhibition of cell division is not sufficient for morphological differentiation. A marked increase in RNA and protein contents of cyclic AMP-induced “differentiated” cells is consistent with an increase in the size of soma and nucleus.     

Envelope synthesis during the cell cycle in Escherichia coli B/r

The rate of synthesis of envelope proteins and phospholipids during the cell cycle of Escherichia coli B/r has been studied using both synchronous cultures and random cultures, first labelled and then subsequently fractionated on an age basis by the membrane elution technique. The rate of total protein synthesis and of phospholipid synthesis, measured by incorporation of [2-³H]glycerol into whole cells, was found to increase exponentially throughout the cell cycle. Total envelope protein was also synthesized continuously throughout the cycle, but the rate of synthesis showed a stepwise pattern with a discrete doubling in rate in the first half of the cycle. Analysis of the pattern of synthesis of about 29 individual envelope polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography revealed that the great majority followed the pattern of the bulk measurements, with a discrete increase in rate of synthesis early in the cycle. One envelope polypeptide, molecular weight 76,000, was, however, only synthesized during a brief period, near the time of division of the bacteria. Pulse-chase studies of envelope polypeptide synthesis in synchronous cultures demonstrated that (1) synthesis and insertion of polypeptide into the envelope was always completed within the pulse period; (2) no post-synthetic modification of polypeptides was detected; (3) one group of polypeptides, including a major outer membrane protein, maintained a stable association with the envelope, whilst a second group displayed considerable “turnover”; (4) about 70% of newly synthesized 76,000 molecular weight protein was lost from the envelope during the succeeding generation.     

Immunity to herpes simplex virus type 2 (HSV-2). I. Development of virus-specific lymphoproliferative and leukocyte migration inhibition factor responses in HSV-2-infected guinea pigs

The development of virus-specific cell-mediated immune (CMI) memory and effector response was studied in strain 13/N guinea pigs infected with herpes simplex virus type 2 (HSV-2) (G). The indirect leukocyte migration inhibition factor (LIF) and the lymphocyte transformation (LT) assays, chosen as probable indicators of effector and memory responses, respectively, were performed simultaneously on spleen cells (SC) obtained at varying times after infection and cultured in the presence of uv-inactivated HSV-2 (G) antigen. Kinetic and dose-response analyses revealed: (i) a time-dependent increase in the magnitude and antigen sensitivity of the LT response as well as a time-dependent decrease in the in vitro “doubling time,” both suggestive of immune maturation, and (ii) a biphasic pattern of LIF production in vitro consisting of an “early” component generated within the first 24 hr in culture, and a “late” component detected between 3 and 6 days in culture. “Late” LIF production correlated well with the lymphoproliferative response and appeared to require the presence of glass-adherent cells and active cell division.     

CELL CYCLE TIME DETERMINATIONS BASED ON LIQUID SCINTILLATION COUNTING OF 3H-TdR LABELED MITOTIC CELLS

Following a 10 min pulse labeling with ³H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G₁, S, G₂ and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.