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.     

TAM selection of Drosophila somatic cell hybrids.

The selection of MDR3, an adenine-salvage-deficient variant of the Kc line, is described. It is resistant to methylpurine and to diaminopurine and is TAM (thymidine, adenine, methotrexate) sensitive. Two wild-type (TAM-resistant) cell lines, Schneider's line 3 (S3) and Dübendorfer's line 1 (D1), due to their different nutritional requirements, are unable to proliferate in medium ZH1% used for line MDR3. This allowed the selection of hybrids between MDR3 and either D1 or S3 in TAM cloning medium after treatment with polyethyleneglycol. Hybrids were identified by the isoenzyme pattern of NADP-dependent isocitrate dehydrogenase.     

Variability of ecdysteroid-induced cell cycle alterations in Drosophila Kc sublines

Abstract. The cell cycle of two lines isolated from Drosophila Kc cells was followed by flow cytofluorometry and cell counting. The first line is the 8-9K clone which grew in a medium supplemented with 5% serum; the second, named subline Kc0, grew in a serum-free medium. The stationary phase is characterized by a G₂ cell accumulation: 73% in the 8-9K clone and 50% in the Kc0 subline.
When the medium was supplemented with the steroid moulting hormone 20-hydroxyecdysone, more than 90% of 8-9K cells and 65% of Kc0 cells were progressively arrested in G₂. In the continuous presence of 20-hydroxyecdysone, most of the 8-9K cells remain G₂-arrested; no massive G₂ release into M was observed and only a few cells were able to divide. When treated for only 3 or 7 days, a transient release into M and proliferation occurred after hormone-free medium renewal, largely masked by G₂ cell death. These results are discussed in comparison with other reports on cell cycle alteration induced by ecdysteroids.     

The differentiation of T-lymphocytes. III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture.

Seven human cultured lymphoblastoid cell lines (CLL) were divided into two major groups based on studies of their cell cycle characteristics and surface Ig. CLL I (lines CL, MW, HH and TM) had generation times ranging from 25–40 hr, S phase times of 10–12 hr, G₂ + M times of 6–8 hr, and demonstrated sharp differences between the percentage of SIg(+) cells in different phases of the cell cycle. Line TM was particularly discordant with the highest percentage of SIg(+) cells in G₂ + M. CLL II (lines PS, JR and HT) demonstrated generation times ranging from 18–21 hr, S phase times of 7–10 hr and G₂ + M phase times of 2 hr. In this second group, two of the three CLLs had no differences between cells taken from different points of the cell cycle. DNA synthesis and cell density could not be correlated with either of the above major parameters, i.e. cell cycle times or SIg expression. The results suggest that human CLLs fall into subgroups in which specific patterns of cellular and immune functions may predominate.     

An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells

The addition of physiological concentrations of the arthropod molting hormone 20-hydroxyecdysone results in the cessation of cell division in the Kc cell line of Drosophila melanogaster. Fluorometric mononitoring of the cell cycle reveals that treatment of the cells with hormone for 12 hr causes a G2 arrest. The dose-response curves are in agreement with those obtained for other hormonal effects in both the Kc line and the intact animal. In the continual presence of hormone, cells remain G2-arrested for approximately 100 hr, resuming division by 120 hr. Cells which have responded once to ecdysteroids and subsequently reentered the cell cycle are insensitive to hormonal restimulation. This lack of response has been correlated with, and is probably due to, the loss of ecdysteroid receptors in stimulated cells.     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

Establishment and characterization of two embryonic cell lines of <Emphasis Type="Italic">Bombyx mori</Emphasis>

Two cell lines, i.e., BmE-SWU1 and BmE-SWU2, were established from silkworm embryonic tissues of the reversion phase through primary culture in Grace’s medium supplemented with 20% fetal bovine serum. The BmE-SWU1 cell line mainly included diploid spindle cells and round cells, which were large and had severe heteroploidy karyotypes. The population doubling time of the 30th passage of the cell line was 58.7 hr. BmE-SWU2 cells were oblong or round, and small. The population doubling time for the 30th passage of the cell line was 46.6 hr. Of BmE-SWU2 cells 89.9% were diploid (2n = 56). Both strains were attached to epithelial-like cell lines and were susceptible to Bombyx mori nucleopolyhedroviruse (BmNPV). Inter simple sequence repeat (ISSR) fingerprinting of silkworm embryonic cell line was obtained.     

Effects of 1,25-dihydroxyvitamin D3 on cell-cycle kinetics of T 47D human breast cancer cells

The replication of several human and animal cancer cell lines is regulated in vitro and in vivo by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonally active form of vitamin D3. We have examined the effects of concentrations of 1,25-(OH)2D3, which inhibit cellular replication, on the cell-cycle kinetics of a 1,25-(OH)2D3-responsive human breast cancer cell line, T 47D. After 6 or 7 days of treatment, a time period representing approximately five cell population doublings of control cultures, concentrations of 1,25-(OH)2D3 in the range 10(-9) M to 10(-6) M caused a time- and concentration-dependent decrease in cell numbers. Treatment of cells growing in charcoal-treated fetal calf serum with 10(-8) M 1,25-(OH)2D3 for 6 days reduced cell numbers to 49% +/- 9% (n = 9) of control, and this was associated with a marked increase in the proportion of cells in the G2 + M phase of the cell cycle from 9.7% +/- 0.5% (n = 11) to 19.6% +/- 2.3% (n = 9), significant by paired analysis (P less than 0.002). At higher concentrations of 1,25-(OH)2D3 (10(-7)-10(-6) M), there was a concentration-dependent decline in S phase and increases in both G0/G1 and G2 + M phase cells. Detailed analysis of the temporal changes in cell-cycle phase distribution following treatment with 2.5 X 10(-8) and 10(-7) M 1,25-(OH)2D3 showed an initial accumulation of cells in G0/G1 and depletion of S phase cells during the first 24 hr of treatment. This decline in S phase cells was not accompanied by a decline in % G2 + M indicating a transition delay in G2 or mitosis. At the lower dose these changes returned to control values at 48 hr and at later times were associated with a slight but consistent decline in G0/G1 phase and an increase in G2 + M. In contrast cells treated with 10(-7) M 1,25-(OH)2D3 had significantly elevated % G0/G1 cells at days 2 and 3, consistent with a transition delay through G1 phase. This was confirmed in stathmokinetic experiments which demonstrated an approximate sevenfold decrease in the rate of exit of cells from G0/G1 following 4 days of exposure to 10(-7) M 1,25-(OH)2D3. This accumulation of cells in G0/G1 was accompanied by a fall in % S phase cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytoplasmic droplets produced by the effect of adenine on Physarum plasmodia: Comparison with caffeine droplets and effect of calcium

Three independently established Drosophila cell lines, Schneider's line 3 (S3), Dübendorfer's line 1 (D1) and MDR3, an adenine salvage deficient clone of the Kc line, all cease to proliferate in the presence of ecdysterone. This is also observed with hybrids between S3 and MDR3 and between D1 and MDR3. It is shown that cells derived directly from wild-type Drosophila embryos can be hybridized with MDR3. Of nine such hybrids all proved to be able to proliferate in the presence of ecdysterone.     

Variability of ecdysteroid-induced cell cycle alterations in Drosophila Kc sublines

The cell cycle of two lines isolated from Drosophila Kc cells was followed by flow cytofluorometry and cell counting. The first line is the 8-9K clone which grew in a medium supplemented with 5% serum; the second, named subline KcO, grew in a serum-free medium. The stationary phase is characterized by a G2 cell accumulation: 73% in the 8-9K clone and 50% in the KcO subline. When the medium was supplemented with the steroid moulting hormone 20-hydroxyecdysone, more than 90% of 8-9K cells and 65% of KcO cells were progressively arrested in G2. In the continuous presence of 20-hydroxyecdysone, most of the 8-9K cells remain G2-arrested; no massive G2 release into M was observed and only a few cells were able to divide. When treated for only 3 or 7 days, a transient release into M and proliferation occurred after hormone-free medium renewal, largely masked by G2 cell death. These results are discussed in comparison with other reports on cell cycle alteration induced by ecdysteroids.     

Ecdysone and the cell cycle: Investigations in a mosquito cell line

Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen.     

CYTOKINETICS OF RAT TRACHEAL EPITHELIUM STIMULATED BY MECHANICAL TRAUMA

Mild abrasion of rat tracheal epithelium results in irreversible damage to the superficial cells and stimulates the viable basal cells to participate in a nearly synchronous wave of DNA synthesis and mitosis. For the growth population as a whole, DNA synthesis started at 14 hr after injury and persisted for 16 hr. The duration of S in individual cells was determined autoradiographically by identifying the time at which a second pulse of DNA precursor (¹⁴C-TdR) was no longer incorporated by cells labelled with ³H-TdR at the onset of S. S was found to be 8–9 hr long. It was also determined that cells entering S at later times synthesized DNA for the same 8–9 hr period. T_G2 was calculated to be 21/2–31/2 hr by subtraction of T_s and 1/2T_M from the period from onset of DNA synthesis to metaphase. By making a second denuding lesion adjacent to the first injury, the cells were stimulated through at least another period of S. At the peak of the second wave of DNA synthesis (50 hr after injury) ¹⁴C-TdR was present in the same cells which had incorporated ³H-TdR administered at the mid-point of the preceding synthetic phase. The 28-hr interval between these two peaks of synthesis is the measure of cell cycle duration for these regenerating tracheal epithelial cells.     

Alterations in radiation induced cell cycle perturbations by 2-deoxy-D-glucose in human tumor cell lines

In the present studies, effects of glucose analogue, 2-deoxy-D-glucose (2-DG) on radiation-induced cell cycle perturbations were investigated in human tumor cell lines. In unirradiated cells, the levels of cyclin B1 in G2 phase were significantly higher in both the glioma cell lines as compared to squamous carcinoma cells. Upon irradiation with Co60 gamma-rays (2 Gy), the cyclin B1 levels were reduced in U87 cells, while no significant changes could be observed in other cell lines, which correlated well with the transient G2 delay observed under these conditions by the BrdU pulse chase measurements. 2-DG (5 mM, 2 hr) induced accumulation of cells in the G2 phase and a time-dependent increase in the levels of cyclin B1 in both the glioma cell lines, while significant changes could not be observed in any of the squamous carcinoma cell lines. 2-DG enhanced the cyclin B1 level further in all the cell lines following irradiation, albeit to different extents. Interestingly, an increase in the unscheduled expression of B1 levels in G1 phase 48 hr after irradiation was observed in all the cell lines investigated. 2-DG also increased the levels of cyclin D1 at 24 hr in BMG-1 cell line. These observations imply that 2-DG-induced alterations in the cell cycle progression are partly responsible for its radiomodifying effects.     

Inhibition of [3H]ponasterone A binding by ecdysone agonists in the intact Kc cell line.

Inhibition of the binding of [3H]ponasterone A ([3H]PoA) by ecdysone agonists including diacylhydrazines such as RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) was examined in intact Drosophila Kc cells. The reciprocal logarithm of the concentration at which there is 50% inhibition of [3H]PoA binding, pIC(50) (M), was determined as the binding activity for all compounds from each concentration-response curve. The order of the activity was PoA>20-hydroxyecdysone>cyasterone>inokosterone>or=makisterone A>methoxyfenozide>or=tebufenozide>ecdysone>RH-5849. The ranking of steroidal ecdysone analogs is consistent with that obtained against Spodoptera Sf-9 cells. Furthermore, in terms of pIC(50), all binding activity for ecdysone analogs, except ecdysone, estimated in the Kc cell line system was significantly higher than that for the Sf-9 cell line system. However, the activity of ecdysone was comparable between Kc and Sf-9 cells. The activity of diacylhydrazine analogs against Kc cells was significantly low compared with that against Sf-9 cells. The potency of methoxyfenozide was 1/200 that of PoA, which showed the highest activity in the Kc cell line system among all compounds tested. The activity of tebufenozide analogs having an n-pentyl or n-hexyl group instead of a 4-ethylphenyl group was similar to that of RH-5849.     

Ecdysteroid-inducible polypeptides in a Drosophila cell line

In the Drosophila melanogaster cell line Kc-H, ecdysteroid hormone treatment causes increased relative synthesis of three ecdysteroid-inducible polypeptides (EIPs), named according to their molecular weights (in kilodaltons) EIP 40, EIP 29 and EIP 28. Increased synthesis of the EIPs is detectable within 45 min (EIP 28) or 75 min (EIPs 40 and 29), is maximal at 4-8 hr and continues for almost 2 days. During this period no other major changes in protein synthesis are discernible using one-dimensional gels. At maximum, EIP 28 synthesis is elevated at least 10 fold above its basal level, and EIPs 40 and 29 somewhat less. EIP induction is ecdysteroid-specific and is detectable in the presence of 10(-8) M 20-hydroxyecdysone. It does not occur in hormone-resistant cells. Apparently identical polypeptides are inducible in another ecdysteroid-responsive cell line, Schneider's line 3. Because EIP synthesis is an early and substantial response to ecdysteroids, this is a promising system for the study of steroid hormone action.     

Cell Cycle Kinetics and Radiation-Induced Chromosomal Aberrations Studied with C14 and H3 Labels

Chinese hamster cells in vitro were double labeled with C(14)TdR and H(3)TdR. At the time of irradiation with Co(60) gamma rays (600 rad), the cells in the G(2) phase were labeled only with C(14), whereas cells in the late and middle S phases were labeled with both C(14) and H(3). The cells in early S phase were labeled only with H(3) and the G(1) cells were unlabeled. Samples were fixed at various time intervals following irradiation and the metaphases were analyzed for chromosomal damage. The phase in which the cell was located at the time of irradiation was determined by counting grains in the first and second layers of autoradiographic film. In both control and irradiated cells some G(1) cells divided prior to some of the cells which were in the S phase denoting mixing of the populations. The G(2) phase sustained three times more chromosomal damage than the S phase. Little difference in chromosomal damage was found between the G(1) and S phases or among the different parts of the S phase. Cells in G(2) sustained a mitotic delay of 4 hr, while the other phases sustained a delay of 2 to 3 hr. Chromatid and chromosome (dicentrics) exchanges were induced in G(1) cells but only chromatid exchanges were induced in S and G(2) cells; this is consistent with the hypothesis that the chromosome consists of two subunits which separate either slightly before or immediately as the cell enters the S phase.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

THE SURFACE FEATURES OF DROSOPHILA EMBRYONIC CELL LINES

Schneider's cell lines 1, 2 and 3 derived from embryonic stages of Drosophila melanogaster were examined by scanning electron microscopy, histochemical staining procedures, and SDS polyacrylamide disc gel electrophoresis. Although the three lines showed some similarities when compared by each of these methods, differences between the lines were observed as well. The surface features of the cells in each line showed morphologic as well as age dependent distinctions when examined in the scanning electron microscope. The intracellular distribution and amount of periodic acid-Schiff positive (PAS+), α-amylase sensitive material was also distinct for each of the lines. Approximately one-third of line 1 cells contained PAS+ hyaluronidase-sensitive material localized within a cytoplasmic vesicle whereas less than 1% of the cells of lines 2 and 3 contained such material. SDS polyacrylamide disc gel electrophoresis revealed PAS+ material in a soluble fraction of line 2 cells and in the 100,000 μ g pellet of line 3 cells, but no PAS+ bands in similar fractions of line 1 cells were detected.     

Novel ceramide analogues display selective cytotoxicity in drug-resistant breast tumor cell lines compared to normal breast epithelial cells.

The sphingolipid ceramide is involved in diverse cell signaling pathways related to proliferation and differentiation. Elevated ceramide also triggers apoptosis. Synthetic ceramide derivatives have been shown to be cytotoxic to tumors, yet few studies have evaluated whether cytotoxicity of synthetic ceramides is selective for tumor cells. We have evaluated the cytotoxic potency of several novel ceramide analogues in the drug-resistant breast tumor cell lines, SKBr3 and MCF-7/Adr, and compared their cytotoxicity in normal breast epithelial cells. Cytotoxicity was assessed using release of lactate dehydrogenase into the culture medium. (2S, 3S)-3-(6'-Dodecylpyridin-2'-yl)-2-butanoylamidopropane-1,3-diol (pyridine-C4-ceramide) produced non-selective cytotoxicity across the three cell types (EC50= 12.8-16.7 microM, at 24 hr). However, 2S,5R-2-(octanoylamido-(3E))-octadecene-1,5-diol (5R-OH-3E-C8-ceramide), (2S,3R)-2-(N-adamantoyl)-(4E)-octadecen-1,3-diol (adamantyl-ceramide), and (2S,3R)-3-(3'-dodecylphenyl)-2-butanoylamidopropane-1,3-diol (benzene-C4-ceramide) exhibited increased cytotoxicity in the tumor cell lines compared to the normal breast epithelial cells. The EC50 values (microM) at 24 hr for these compounds in SKBr3 cells, MCF-7/Adr cells, and normal breast epithelial cells, respectively, were as follows: 5R-OH-3E-C8-ceramide, 18.3, 21.2 and 58.7; adamantyl-ceramide, 10.9, 24.9 and >100; benzene-C4-ceramide, 18.9, 45.5 and >100. At a concentration of 30 microM, the fold increase in cytotoxicity in breast tumor cell lines compared with normal breast epithelial cells was as follows: 5R-OH-3E-C8-ceramide, 23.7 and 19; adamantyl-ceramide, 11.2 and 10.3 and benzene-C4-ceramide, 79.3 and 77.2, for SKBr3 and MCF-7/Adr cells, respectively. Possible mechanisms accounting for selectivity are discussed. Ceramide analogues with relatively selective toxicity against tumor cells may have potential as therapeutic agents. Elucidating the mechanisms of selective cytotoxicity could identify novel targets that may lead to development of anti-neoplastic agents with a higher therapeutic index.     

Double labeling with iodo- and bromodeoxyuridine for cell kinetics studies

The rate of progression through the cell cycle was determined in five human glioma cell lines by a new sequential immunohistochemical staining technique. The cells were labeled first with iododeoxyuridine (IdUrd) for 1-3 hr and then with bromodeoxyuridine (BrdUrd) for 30 min. Labeled cells were identified with Br-3, a monoclonal antibody that recognizes only BrdUrd, and with IU-4, an antibody that recognizes both IdUrd and BrdUrd. Each slide was stained sequentially, first with the immunoperoxidase method for Br-3 and then with the alkaline phosphatase-anti-alkaline phosphatase method for IU-4. Cells that were positive only for IU-4 represented the fraction of S-phase cells that passed into the G2 phase during the period of incubation with IdUrd. The rates of progression measured by this method were constant in each cell line and resulted in smaller standard errors than were obtained by measurements from specimens stained singly for IdUrd and BrdUrd in different slides. The duration of the S-phase calculated from this fraction in the five cell lines ranged from 8-13 hr; the estimated potential doubling times were 25-32 hr and were very similar to the actual doubling times.