pH dependency of cell attachment and growth at both clonal and subculture densities of cultured lepidopteran cells

Summary TN-368 cells were seeded at 10⁶ per flask in TNM-FH medium adjusted to a variety of pH levels which ranged from approximately 5.9 to 6.8. In general, growth was similar from pH 6.2 to nearly 6.7. The medium pH increased with time in culture to a maximum near 7.0 for all pH levels. Similar results for growth and pH increase were also obtained when the cells were plated at densities of 10⁴ and 10⁵ per flask. Both the fraction of attached cells and the relative intensity of attachment increased with seeding pH. Cells seeded near pH 6.7 or above frequently required vigrrous procedures such as trypsinization to detach them. DNA synthesis was measured and found to be similar for cells seeded in medium between pH 6.2 and 6.7. Colony forming efficiency increased from approximately 27% at pH 5.9 to 39% at 6.2, remained in the region of 40% between 6.2 and 6.7 with a peak of 48% at 6.6, and plunged abruptly to a few percent just above 6.7 and was near zero above 6.8. Colony morphology was optimal near pH 6.6. This work was supported by USPHS grant R01 CA34158, awarded by the National Cancer Institute, DHHS, Bethesda, MD.     

Establishment and characterization of a cell line derived from bovine tracheal glands

Summary Bovine tracheal submucosal gland cells have been isolated by enzymatic digestion and serially propagated in tissue culture for more than 12 mo. (40 passages). The cells exhibit an epithelioid appearance at confluence and contain alcian blue (pH 2.5)/periodic acid-Schiff-positive material within cytoplasmic granules. By electron microscopy numerous osmiophilic secretory granules are seen. Maximal growth is observed when the cells are grown on human placental collagen-coated culture vessels in medium supplemented with 20% fetal bovine serum. Scintillation spectrometry revealed that radiolabeled precursor (³⁵SO₄) was incorporated into high molecular weight molecules and released from cells. Isoproterenol (10⁻⁶ to 10⁻³ M) stimulated the release of³⁵SO₄. The maximal response to isoproterenol was completely inhibited by the β-adrenergic antagonist propranolol. It is concluded that the cultured cells retain features of tracheal gland cells and may serve as a useful model of synthesis and secretion of macromolecules by tracheal gland cells. This study was supported in part by NIH Program Project grant HL-24136, by a National Cystic Fibrosis Foundation Research Development Grant, and by a grant from Cystic Fibrosis Research, Inc. Dr. Finkbeiner is a recipient of NIH Clinical Investigator Award HL-01387.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING APPLICATION OF A SKIN IRRITANT (CANTHARIDIN)

The strong skin irritant cantharidin dissolved in benzene was applied to the back of hairless mice. Single cell suspensions of epidermal basal cells were obtained and flow microfluorometric measurements of cellular DNA content were made. Smears were made for autoradiography, and the [³H]TdR labelling index (LI) and mean grain count (MGC) were assessed up to 3 days after cantharidin application. Three successive peaks of cells with S phase DNA content accompanied by three LI peaks were observed. The first two peaks were follwed by peaks of cells in G₂ phase, indicating that after the acute cell injury caused by cantharidin the cells traversed the cell cycle in partial synchrony through two subsequent cell cycles, each of 10–12 hr duration. During this phase of rapid proliferation the LI reached the proportion of cells in S phase, contrary to what is observed in untreated mouse epidermis, where the labelled cells contribute to about half the proportion of cells with S phase DNA content. The first two peaks of cells in S phase and LI coincided with an increased MGC, whereas the third peak was accompanied by a MGC significantly below control values. This indicates that this latter peak is due to a longer DNA synthesis time rather than to a partially synchronized and increased cell proliferation. The duration of the G₁, S and G₂ phases seems to be reduced initially in rapidly proliferating epidermis.     

G-Protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca²⁺ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 M GTP-S did not significantly affect prepulse facilitation or whole-cell Ba²⁺ current (I _Ba) density. In contrast, inactivation of G proteins by intracellular GDP-S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of I _Ba, respectively. GDP-S dialysis resulted in nearly a threefold increase in peak I _Ba density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 m intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca²⁺ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (50%) in I _Ba amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca²⁺ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.This work was supported by a National Science Foundation grant (DCB-8812562), American Heart Association-Ohio Affiliate grant (SW-91-18), and an American Parkinson's Disease Association grant. C.A.D. was supported by a predoctoral National Research Service Award (National Institutes of Health training grant HL07571-08). The authors thank Kluener's Packing Co. for their generous supply of adrenal glands.     

Unit gravity sedimentation analysis of basic nuclear protein synthesis during the HeLa cell cycle

Summary Exponentially growing HeLa cells have been separated according to their cell cycle age by sedimenting at unit gravity for 3 hr on a phosphate-buffered sucrose density gradient. Measurements of cell size, cell number, DNA content, and tritiated thymidine incorporation in consecutive portions of the gradient showed that cells in upper fractions were in G₁, cells in middle fractions were in S, and cells in lower fractions were in G₂. Basic amino acids were rapidly incorporated into nuclear protein during late G₁ and S; some incorporation also took place during G₂. This work is supported by grant A-3458 from the National Research Council of Canada.     

Preparation and characterization of rabbit myometrial cells in primary culture: Influence of estradiol and progesterone treatment

Summary Myometrial cells were obtained following a three-step enzymatic digestion of uterine horns from Day 1 pseudopregnant rabbits. Isolated cells were cultured in PRMI 1640 supplemented with 10% fetal bovine serum (FBS), whole or steroid depleted (FBS-DC) at a plating density of 0.5×10⁶ cells/ml. The cells reached confluency on Day 6 to 7 with whole serum and on Day 7 to 8 with DC serum. The process yielded myometrial cells at a purity level of at least 80% as assessed by indirect immunofluorescence using desmin antibody on confluent cultures. The addition of increasing doses of 17β-estradiol (E₂) (0.1 nM to 1 μM) to the culture medium resulted in an increase in total protein and DNA content (1.5-fold at 1 nM). Similar treatment with progesterone (P) resulted in a 25% inhibition of protein and DNA content at 10 nM. Pretreatment of cells with E₂ (1 nM) for 3 d followed by P (10 nM) for 3 d resulted in a 1.8-fold stimulation of protein with a higher protein: DNA ratio indicating that the increase was due to cellular hypertrophy. Analysis of desmin by polyacrylamide gel electrophoresis showed that this cytoskeleton protein was not affected by steroid treatment. Our results indicate that PR can generate two different responses depending on cell pretreatment. In as much as myometrial cells grown in primary culture respond differentially to E₂ and P they should provide a useful model to study the regulation of myometrial contractility. This research was supported by Natural Sciences and Engineering Research Council of Canada, Ottawa, Ontario, grant no. U-0389.     

Synchronization of drosophila cells in culture

Summary We synchronized Drosophila cell lines (Schneider's line 2 and K_c) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S=14 to 16 hr; G₂=6 to 8 hr; M=0.4 hr) were similar to those of K_c cells. This work was performed under the auspices of the U.S. Energy Research and Development Administration. A.R. was supported by an NIH post-doctoral fellowship, No. CA01060.     

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Induction of DNA Synthesis and Cell Division by Isoleucine and Glutamine in G1-Arrested Cells in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G₁ following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G₁, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10^-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 10⁵ cells/ml. Under similar conditions, about 4 x 10^-6 M isoleucine was required for all G₁-arrested cells to progress through cell division. In contrast, 1 x 10^-4 M glutamine was necessary for maximum initiation of DNA synthesis in G₁ cells, along with sufficient isoleucine. A technique for rapid production of G₁-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G₁ after 30 hr.     

Responses of type II pneumocyte antioxidant enzymes to normoxic and hyperoxic culture

Summary Cultured type II pneumocyte responses to in vitro normoxia (95% air: 5% CO₂) or hyperoxia (95% O₂:5% CO₂) were quantified. Normoxic culture (0 to 96 h) of rabbit type II cells resulted in enhanced cell-monolayer protein and DNA content. During this same time, cellular activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH Px) decreased. Compared to cultures maintained in normoxia, hyperoxic exposure of cultures resulted in decreased cell-associated protein and DNA content. Exposure to hyperoxia also resulted in cytotoxicity as demonstrated by elevated cellular release of DNA, lactate dehydrogenase (LDH), and preincorporated 8-[¹⁴C]adenine. Cellular catalase and GSH Px activities in hyperoxic cells decreased similarly to normoxic controls. In contrast, cellular SOD activity in hyperoxic cells decreased less than in normoxic cultures. Cellular SOD activity in hyperoxic cultures, when normalized for cellular protein, but not DNA, was greater than normoxic values after 24 to 96 h of exposure. Unlike the decrease in cellular antioxidant enzymes during normoxic and hyperoxic culture, cellular LDH activity increased during both these exposures. Cellular LDH activity in 24 to 96 h hyperoxia-exposed cells increased to a lesser extent than normoxic controls. The extent of depression in LDH activity was dependent on whether the activity was normalized for cellular protein or DNA. Type II pneumocytes, which normally undergo hyperplasia and hypertrophy during hyperoxia in vivo, exhibited oxygen sensitivity in vitro. Exposure of type II cells to hyperoxia in vitro resulted in alterations in cellular SOD and LDH activities, but recognition of such changes were dependent on whether enzymatic activities were normalized for cellular DNA or protein. This work was supported by a grant from the Health Effects Institute, grant HL40458 from the National Institutes of Health, Bethesda, MD, and a grant from the American Lung Association, New York, NY.     

RNase-sensitive DNA polymerase activity in cell fractions and mutants of Neurospora crassa

RNase-sensitive DNA polymerase activity (RSDP) was tested in different cell fractions of Neurospora crassa cell types and its morphological mutants. This RSDP was found localized in the microsomal pellet fraction and absent in the purified nuclear pellets isolated from different N. crassa cell types: conidia, germinated conidia, and mycelia. This enzyme is capable of synthesizing a DNA product only in the presence of all four deoxyribonucleoside-5-triphosphates and Mg²⁺. Removal of RNA from the pellet fraction by RNase strongly inhibited the DNA synthesis. The endogenous synthesis of DNA in the microsomal pellet fraction was associated with the formation of an RNA:DNA hybrid as analyzed by Cs₂SO₄ equilibrium density gradient centrifugation. The DNA product after alkali hydrolysis hybridizes with the RNA isolated from the same pellet fraction, as analyzed by elution from hydroxylapatite column at 60 C. This DNA product did not hybridize with poly(A). A few mutants tested showed this RNase-sensitive DNA polymerase activity.This work was supported in part by a contract with the U.S. Department of Energy and a grant from the U.S. Naval Research.     

Buoyant densities of DNA of mammals

One characteristic of DNA, CsCl buoyant density peak values, was determined for DNA samples isolated from 93 species belonging to 11 orders of mammals. The CsCl buoyant density values varied over a very narrow range, 1.696–1.701 g/cm³. Satellite DNAs were found in a number of species. The function and origin of these satellite DNAs are not known.This work was supported by grant DRG-269 from the Damon Runyon Memorial Fund for Cancer Research, Inc., and GB-6657 from the National Science Foundation.     

Alterations of the incorporation of [3H]thymidine in cells in culture regulated by nucleoplasmic extract

Porcine skin nucleoplasmic extract (PSNE) was shown to alter the incorporation of [³H]thymidine into DNA of selected porcine, bovine, and human cell populations in culture. PSNE stimulated incorporation of [³H]thymidine into DNA of porcine and bovine dermal cells an average of 300 and 200% of control value, respectively. When porcine and bovine epidermal cells were exposed to PSNE the treatment inhibited [³H]thymidine incorporation into DNA by an average of 48 and 45%, respectively. Similar inhibitions were observed for porcine and bovine kidney, porcine lung, and human KB cells. Thus, the effect of PSNE on the incorporation of [³H]thymidine into DNA of various cultured cells was either stimulatory to dermal cells or inhibitory to a variety of other cell types, including skin epidermal cells. The stimulatory and inhibitory effects of PSNE were abolished by heating PSNE for 5 min in boiling water before its addition to cell cultures. This suggests that macromolecular structure is important in the action of PSNE. This project was supported by a grant from the Research Advisory Board, University of Nevada, Reno, NV.     

Studies on the isolation and purification of chloroplasts from Euglena gracilis

Summary A method has been developed for the isolation of chloroplasts from Euglena gracilis grown under mixotrophic conditions. This method utilizes sucrose density-gradient centrifugation in the AXII zonal rotor and allows the rapid preparation of large amounts of chloroplasts free from contaminating whole cells and other cytoplasmic materials. The majority of the isolated chloroplasts appear intact in phase contrast and electron micrographs. The purified chloroplast fraction contains DNA, the major species of which has a density of 1.682 g/cm³. The species of DNA having a density of 1.707 g/cm³ seemed to result from the presence of contaminating nuclear fragments which could be removed by isopycnic flotation.This research was supported in part by a grant from USPHS No. AM-07189 (to J. M. E.), American Cancer Society Fellowship No. PF-443 (to J.F.P.) and grant No. 2972E06 from the Office of Sponsored Research, University of Florida (to J.F.P.). Univ. of Fla. Agr. Exp. Stat. No. 4538.     

S phase synchrony in monolayer CHO cultures

Parameters are described for reproducible S phase synchrony of Chinese hamster ovary cells growing in monolayer, adapting a method described by Tobey & Crissman [1] for CHO cells growing in suspension culture. Cells are collected at the G1/S boundary in hydroxyurea after reversal of an early G1 block induced by isoleucine deprivation. The entire population enters the S period within 60 min after removal of hydroxyurea and proceeds through the S period with minimal decay of synchrony, as evidenced by autoradiographic and rate studies on [³H]TdR uptake. In addition, a method is described for obtaining cells synchronized during two successive S periods. The presence of hydroxyurea during G1 does not measurably affect the rate of uptake of [³H]uridine or [³H]leucine into TCA-insoluble material; however, cultures released from the hydroxyurea block at 10 h incorporate slightly more [³H]uridine (but not [³H]leucine) in the next 6 h than cultures maintained in hydroxyurea over this interval. Delaying entry into S with hydroxyurea for as long as 15 h does not significantly change the initial rate or duration of DNA synthesis upon removal of hydroxyurea, arguing against the build-up of substances responsible for initiation of replicons. Furthermore, if DNA synthesis is delayed with hydroxyurea in one cell cycle, a constant minimal interval of 15 h elapses before the population enters into the next S phase, suggesting that the timing of the S period is coupled to the timing of the previous S.     

Na+-independent l-alanine uptake by trout cells. Evidence for the existence of at least two functionally different asc systems

The Na⁺-independent uptake of l-alanine has been studied in trout red blood cells, isolated hepatocytes and peripheral blood lymphocytes. The present study shows the existence of two functionally different Na⁺-independent systems for short chain neutral amino acids in these cells. They are designated as asc systems because of their resemblance to systems described in other cell types. Besides their independence of sodium and a rough similarity in substrate preference, the most important property shared by the two carriers is a lack of trans-stimulation, allowing further differentiation from system L. One of them is an unusually stereospecific carrier present in red blood cells, the other is less restrictive and present in hepatocytes and peripheral blood lymphocytes. Extracellular acid pH increases the incorporation to red blood cells, while it slightly depresses the uptake in the other cells. From the data presented, it is not possible, at first, to classify these carriers as asc ₁ or asc ₂ systems. Moreover, the system present in red cells resembles that found in the nonerythroid cells, BSC-1, while there is no clear parallelism between the system found in hepatocytes/lymphocytes and any of those described previously.This work was supported in part by a grant from the DGICYT (PB91-0235) of the Spanish Government and by a grant from the CIRIT (AR91-21) of the Generalitat de Catalunya. M.A.G. is a recipient of a fellowship from the Generalitat de Catalunya. We would like to express our sincere thanks to Mrs. Rosa Marsol and Mr. Antonino Clemente (Medi Natural, Generalitat de Catalunya) for their help and logistical assistance and to Mr. Robin Rycroft for his editorial help. J.L. Albi, P. Canals and M.A. Gallardo contributed equally to this study.     

Pharmacokinetics of anti-ganglioside GD2 mAb 14G2a in a phase I trial in pediatric cancer patients

A phase I trial of a murine anti-ganglioside (GD2) monoclonal antibody (mAb) 14G2a was conducted in 14 neuroblastoma patients and 1 osteosarcoma patient to assess its safety, toxicity and pharmacokinetics in pediatric patients. The pharmacokinetics of mAb 14G2a were biphasic with at ₁ ^2/ of 2.8±2.8 h and at ₁ ^2/ of 18.3±11.8 h. In general,t ₁ ^2/ was dose-dependent with a level of significance ofP=0.036, and it reached a plateau at doses of 250 mg/m² or more. Overall the peak serum levels were dose-dependent atP<0.001. However, they demonstrated an abrupt increase between doses of 100 mg/m² and 250 mg/m². The latter two suggest a saturable mechanism for mAb elimination. In addition, peak serum concentrations were observed earlier at higher mAb doses, which indicates the achievement of a steady state. Thet ₁ ^2/ of mAb 14G2a in children appears to be shorter than in adults. Furthermore, 2 patients demonstrated a considerable decrease int ₁ ^2/ following retreatment with 14G2a. This was paralleled by high human anti-(mouse Ig) antibody levels. This study represents the first comprehensive analysis of murine mAb pharmacokinetics in children and will be useful in the future design of mAb therapy.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439 and in part by a grant from the general Clinical Research Center program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M. M. U.-F. and C.-S. H. were supported in part by a grant from the Children's Cancer Research Foundation, and R. A. R. was supported in part by NIH grant CA 42508     

Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line,RTG-2

Summary Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures. Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After 3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined. This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation should allow the G₁-S transition to be studied in a representative of teleosts. This research was supported by Natural Sciences and Engineering Research Council of Canada grant to N. C. B.     

In vitro host range of the Hz-1 nonoccluded virus in insect cell lines

A total of 13 insect cell lines spanning 4 orders (Lepidoptera, Coleoptera, Diptera, and Homoptera) were tested for their ability to replicate the nonoccluded virus Hz-1. Only the Lepidopteran cell lines supported replication of the virus with TN-CL1 and BCIRL-HZ-AM1 producing the highest titers of 2.4 × 10⁸ tissue culture infective dose (TCID)₅₀/ml and 2.0 × 10⁸ TCID₅₀/ml, respectively. A codling moth cell line (CP-169) was the only Lepidopteran cell line that did not replicate the virus and transfection of this cell line with Hz-1 DNA failed to replicate the virus. Also, transfection with DNA from a recombinant baculovirus carrying the red fluorescent protein gene (AcMNPVhsp70 Red) was not expressed in CP-169 cells. The replication cycle of Hz-1 in BCIRL-HZ-AM1 cells showed that this virus replicated rapidly starting at 16 h postinoculation (p.i.) and reaching a peak titer of 1.0 × 10⁸ TCID₅₀/ml 56 h postinoculation. Hz-1 when compared with several other baculoviruses has the widest in vitro host spectrum.     

Biological heterogeneity and radiation sensitivity of in vitro propagated lung metastatic lines originated from a transplantable squamous cell carcinoma of BALB/C mouse

Summary Seven cell lines established from a diethylnitrosamine (DEN)-induced forestomach carcinoma (DEN₃) of a BALB/c mouse and its six pulmonary metastatic foci were used to study the biological and functional diversity of tumor cells. DEN₃ is a highly tumorigenic line capable of forming lung metastases readily. Six metastatic nodules were isolated from the lungs of syngeneic mice and six cell lines were established. The cell lines differed in characteristics such as tumorigenicity, metastatic capability, and in vivo and in vitro growth properties. Radiation sensitivity of these cell lines was examined by exposure, at ner confluency stage of in vitro growth, to doses of 2.5 to 50 Gray (Gy) X-rays (1 Gy=100 rads). Shortly after exposure (approximately 5 min), the cells were harvested and 10⁵ cells were cultured or inoculated into syngeneic mice, or both. Growth of three of the six cell lines tested was prohibited by 5 Gy. However, some populations from the other cell lines were able to survive 5 or 10 Gy. Progenies of the cells that survived primary radiation exposure after several in vitro passages were able to withstand another exposure of the same magnitude but not a higher dose. The X-rayed survivor cells also maintained their tumorigenic potential. This work was supported by Biomedical Research Support grant 2-507-RR-071292-07 from the National Institute of Health, Bethesda, MD; by the Ohio Board of Regents Research Challenge Grant; and an FRC grant. R. J. Jamasbi under Department of Energy Research contract DE-ACOF-760-R00033 through Oak Ridge Associated Universities.     

NEW METHODS OF CELL CYCLE ANALYSIS BASED ON THE SELECTIVE TAGGING OF CELLS EITHER AT THE BEGINNING OR END OF S PHASE

New techniques for cell cycle analysis are presented. Using HeLa cells, methods are described for the selection of a narrow window or cohort of lightly [³H]-labeled cells located either at the very beginning or the very end of S phase. The cohort cells are tagged by a labeling procedure which entails alternating pulses of high and low levels of [³H]thymidine and are identified autoradiographically. Additional methods are described for following the progress of cohort cells through the cell cycle. Theoretically, with the methods described, it should be possible to follow the ‘early S cohort’ cells as they exit from S phase, as they enter and exit M and as they enter the subsequent S phase. This would allow a determination of S, S + G₂, S + G₂+ M and T. It should theoretically be possible to follow ‘late S cohort’ cells in a similar manner, allowing a determination of G₂, G₂+ M and G₂+ M + G₁. To test these predictions, several experiments are presented in which the progress of the two cohorts is monitored. The best data were obtained from the mitotic curves of cohort cells. For each of the cohorts, values were obtained for the time required for peak concentration of cells in mitosis, the coefficients of variation and of skew. The curve of cohort cells passing through mitosis is shown to fit a log-normal curve better than a normal curve. In addition, the mitotic curves are used to estimate the length of M and to estimate the loss of cohort synchrony. Other uses of these methods are discussed.