Methotrexate permits a limited number of cell doublings and affects mitochondrial substructure inTetrahymena

Summary Methotrexate (MTX), a folic acid analogue, is used in cancer chemotherapy in low or high doses. Addition of MTX to proliferatingTetrahymena cultures revealed that, irrespective of the concentration of MTX (1–50 mM), the cells doubled once during a 6-hour exposure and twice during a 24-hour exposure; the normal generation time is 3 hours. MTX had a dose-dependent effect on the maximal number of cell doublings reached after 72 hours, thus 5 1/2 and 3 1/2 doublings were found in 1 and 10 mM MTX, respectively. Massive cell death was observed after a 4-day exposure. At all concentrations of MTX, the rate of endocytosis was unaffected initially but decreased to 60% of the control value after 24 hours. Conspicuously, small refractive, or electron dense, granules accumulated in MTX-treated cells; these granules are presumably lysosomes accumulating MTX for defecation. The mitochondrial substructure became altered in MTX-treated cells; after a 24-hour exposure to MTX the mitochondria were almost depleted of tubules. This reduction of the number of mitochondrial tubules occurred gradually, apparently correlated with division of the organelles. Recovery was also studied of cells exposed for 1 or 3 hours to 10 mM MTX. During the first 24 hours after removal of MTX, the cells were affected in a manner similar to that described for cells exposed continuously to MTX, also with respect to the altered substructure of mitochondria; however, the recovering cells resumed the normal rate of cell proliferation after 4 cell doublings. These longlasting effects, of even a short exposure ofTetrahymena to MTX, may in part explain the cause of the severe side effects accompanying MTX chemotherapy.     

Radiosensitizing effect of hyperfractionated radiation

To determine whether hyperfractionated treatment has benefits in the radiation therapy, two melanoma cell lines were irradiated with eight 0.5 Gy fractions as well as one single 4 Gy in vitro. The radiation was performed in air and in hypoxia as well. Cells were also irradiated in the presence of dibromodulcitol, a bifunctional alkylating agent with a weak radiosensitizer effect. The aim of the study was to examine whether hyperfractionation can influence the radiosensitizing effect of the bioreductive agent. Survival of the cells was determined immediately and 24 hours after various treatments by cell counting in hemocytometer and clonogenic assay. The number of the apoptotic cells was determined by the TUNEL assay and was followed up to 72 hours after treatment. Hypoxic cells had higher sensitivity than normoxic cells after 0.5 Gy irradiation. Radiosensitizing enhancement of DBD was higher with fractionated irradiation. The number of the apoptotic cells was significantly higher after hyperfractionated treatments than after single dose treatment combinations. Our results showed the significance of the hyperfractionated irradiation with 0.5 Gy per fraction in vitro.     

Induction of the replicative synthesis of DNA by SV40 virus T antigen introduced into cells using liposomes

The role of virus SV40 T-antigen in the induction of cell DNA synthesis during its incorporation into cell liposomes was studied, using monolamellar liposomes obtained by phase reversal with incorporated highly purified T-antigen. Immunofluorescence studies revealed that T-antigen effectively penetrates inside the cells and after 10 hours is accumulated in the nuclei, where its level remains unchanged for 24 hours. Injections of purified T-antigen into the renal cells of serum-starved CV1 monkeys resulted in an almost 10-fold increase in the number of DNA-synthesizing cells 18 hours after the exposure. The same effect was observed during stimulation of a 10% serum culture. Removal of T-antigen from the preparation by specific immunoadsorption eliminated this effect. Centrifugation of cells grown in the presence of bromodeoxyuridine in a CsCl gradient was used to demonstrate the replicative type of cell DNA synthesis during T-antigen induction.     

HL-60-AR白血病细胞诱导分化性状的持续性表达

HGPRT~-人早幼粒白血病细胞突变株(HL-60-AR)与RA保温一定时间后,洗去药物继续培养,细胞分化性状(NBT还原能力、细胞膜C_3补体受体及形态变化)不但继续存在,而且能持续表达。撤去RA后连续传代培养,至少在传三代后细胞分化性状仍高度表达。然而,DMSO对HL-60-AR细胞的作用特点明显不同于RA。HL-60-AR细胞分化伴随增殖能力的降低。核酸分子杂交结果表明,细胞c-myc癌基因表达受抑先于细胞分化性状的获得和增殖能力的下降。     

Progenitor cell cycling during hair cell regeneration in the vestibular and auditory epithelia of the chick

We investigated nucleotide-labeling patterns during ongoing hair cell regeneration in the avian vestibular epithelium and during drug-induced regeneration in the avian auditory epithelium. For utricle experiments, post-hatch chicks received an injection of bromodeoxyuridine (BrdU) and were allowed to survive from 2 hours to 110 days after the injection. Utricles were fixed and immunoreacted to detect BrdU. The number of BrdU-labeled nuclei in the hair cell and support cell layers of the utricular sensory epithelium changes significantly between 2 hours and 110 days post-BrdU. At 2 hours, most labeled cells are isolated, while by 5–10 days, the majority of labeled cells are organized in pairs that are most frequently composed of a hair cell and a support cell. Pairs of labeled cells are seen as late as 110 days. Clusters of more than 3 labeled cells are uncommon at all time-points. The total number of labeled cells increases approximately 1.5-fold between 5 and 60 days post-BrdU. This increase is due primarily to a rise in the number of labeled support cells, and it is likely that it represents additional rounds of division by a subset of cells that were labeled at the time of the BrdU injection. There is a significant decrease in labeled nuclei in the hair cell layer between 60 and 110 days post-BrdU, suggesting that hair cells die during this period. To investigate support cell recycling in the drug-damaged auditory epithelium, we examined nucleotide double labeling after separate injections of BrdU and tritiated thymidine. A small number of support cells that incorporate BrdU administered at 3 days post-gentamicin treatment also label with tritiated thymidine administered between 17 and 38 hours later. We conclude that a small population of support cells recycles during regeneration in both the normal utricle and the drug-damaged basilar papilla.     

Early administration of methylprednisolone decreases apoptotic cell death after spinal cord injury

The purpose of this study is to evaluate, in an experimental model of spinal cord injury (SCI), the presence of apoptotic cell death after trauma and if early administration of a single bolus of methylprednisolone (MP) influences apoptosis in the zone of trauma and in adjacent spinal cord segments. For this study, a total of 96 adult female Wistar rats were subjected to spinal contusion at the T6-T8 level, producing immediate paraplegia. Forty-eight animals (treated group) received a single intraperitoneal injection of MP, at a dose of 30 mg/kg body weight, 10 minutes later. Cells undergoing apoptosis were detected by means of immunohistochemical labeling with the monoclonal antibody Apostain (anti-ssDNA MAb F7-26), in the injured spinal cord tissue, both in the zone of the lesion and in the adjacent spinal segments (rostral and caudal zones), 1, 4, 8, 24 and 72 hours and 1 week after injury. Apoptosis was detected in neurons and glial cells in the zone of the lesion 1 hour after trauma, with a pattern that showed no changes 4 hours later. Between 4 and 8 hours postinjury, the number of apoptotic cells increased, after which it decreased over the following days. In the adjacent spinal segments, apoptotic cells were detected 4 hours after trauma, and increased progressively over the remainder of the study, the number of apoptotic cells being similar in the lesion zone and in rostral and caudal zones one week after injury. When the group of MP-treated animals was considered, significant decreases in the number of apoptotic cells were detected in the lesion zone 24 hours after injury, and in the rostral and caudal zones, at 72 hours and at 1 week after trauma. These findings show that early administration of a single bolus of MP decreases apoptotic cell death after SCI, supporting the utility of MP in reducing secondary damage in injured spinal cord tissue.     

Effects of colchicine on the morphology and prolactin secretion of rat anterior pituitary cells in monolayer culture

The effects of incubation of rat anterior pituitary cells in monolayer culture with 10(-6) M colchicine have been investigated during time-intervals extending from 1 to 96 hours. Prolactin release, as measured by radioimmunoassay, was rapidly inhibited by colchicine, this inhibition being accompanied by increased cellular prolactin content for up to 24 hours of treatment and followed by decreased values of cellular prolactin concentration at later time-intervals. Immunocytochemical localization showed an increased positive reaction for prolactin up to 24 hours after colchicine treatment, whereas transmission electron microscopy demonstrated, in parallel, an increased number of intracellular prolactin secretory granules during the same interval. Longer periods of treatment (24-96 hours) resulted in the appearance of more lysosomes, autophagic vacuoles and microfilaments in the cells, whereas the number of Golgi elements was decreased. Following four hours of colchicine treatment and at later stages, microtubules could no longer be observed in the sections. Scanning electron microscopic data showed that colchicine treatment induced dramatic changes in the cell surface morphology: at short time intervals (4 and 8 hours), the number of microvilli decreased and the cell surface became folded, whereas, later, "bleb"-like protrusions of variable dimensions partially covered the cell surface and seemed to be released from it. These data show a good correlation between secretory activity of prolactin-producing cells and morphological changes induced by colchicine treatment.     

Stimulation of DNA synthesis in cultures of mouse embryo fibroblast-like cells

Stimulation of the DNA synthesis and mitoses in stationary cultures of mouse embryo fibroblast-like cells was induced by various agents such as ribonuclease, digitonin, fresh medium and commercial preparations of hyaluronidases. Time sequence of stimulation was similar in experiments with all these agents. Cells were activated to enter S phase from GI phase. The rise of the number of DNA-synthesizing cells was preceded by a latent period of about 8–12 hours with the maximal number of DNA-synthesizing cells being observed at 16–24 hours. Mitotic wave was observed after the wave of DNA synthesis. Stimulation of DNA synthesis and mitosis was not preceded by any significant decrease of an average cell density in the culture. The progeny of activated cells had no greater chance than other cells to be activated again when stimulation was repeated. It is concluded that similar proliferative reactions can be induced in stationary cultures by a variety of diverse agents. Possible role of cell surface changes in the induction of these reactions is discussed.     

硫化砷对Jurkat细胞增殖的抑制作用及其机制

目的：研究硫化砷（As4S4）作用于人类急性T细胞白血病细胞株Jurkat细胞后对其增殖的影响及机制。方法：用不同浓度硫化砷（0,2．5,5,10,20txM）作用于Jurkat细胞不同时间（24,48,72小时）,通过四甲基偶氮唑蓝（MTT）法观察对其增殖的抑制作用。倒置显微镜下观察不同浓度硫化砷（0,5,10,20txM）作用Jurkat细胞24小时后细胞数目和形态变化。利用Westemblot方法检测硫化砷（0,5,10uM）作用Jurkat细胞24小时后胞内CleavedNotchl蛋白和C．myc蛋白变化情况。结果：@MTT结果提示硫化砷在一定浓度范围内对Jurkat细胞有明显的抑制作用（P〈0．05）,并呈浓度和时间依赖性。②显微镜下观察到硫化砷处理Jurkat细胞24小时后,细胞数目随药物浓度增加而减少,而其细胞碎片随着药物浓度增加而增加。@Westernblot结果显示硫化砷处理24小时后,胞内CleavedNotchl蛋白、C-myc蛋白下调,并随浓度增加下降的更为明显。结论：硫化砷对Jurkat细胞生长具有抑制作用．其机制可能通过影响Notch信号通路起作用。     

AN ELECTRON MICROSCOPE STUDY OF TOBACCO MOSAIC VIRUS LESIONS IN NICOTIANA GLUTINOSA L

Ultra-thin sections of Nicotiana glutinosa L. leaves inoculated with a concentrated solution of tobacco mosaic virus were made at short intervals from 0 to 78 hours after inoculation. Eight hours after inoculation, the size of starch grains increased. This was followed by rupture of cytoplasmic and chloroplast membranes. At about 24 hours there was a great increase in number of mitochondria, which persisted until about 60 hours, when some became electron opaque while others appeared to disintegrate. Finally, the cell contents were compressed into one area of the cell, where they became electron opaque. This was accompanied by collapse of the rest of the cell and tearing away of the cell walls from adjacent cells. The nucleus remained stable and intact for as long as observations could be made. No identifiable virus particles were seen.     

Correlation of HSP110 expression with all-trans retinoic acid-induced apoptosis

In a previous study, we observed the strong expression of a stress protein of the HSP100/Clp family (HSP110) in apoptotic mesectodermal cells during early mouse facial development. In the present study, we describe the strong expression of the same HSP110 in mesectodermal cells undergoing apoptosis after all-trans retinoic acid (RA) administration. We used a teratological model known to increase cell deaths mainly in the first and second branchial arches during mammalian cephalogenesis: the treatment of E9 mouse embryos with all-trans RA, which results in craniofacial malformations comparable to those that characterize mandibulofacial dysostosis in man. Pregnant NMRI mice were treated with 60 mg/kg body weight of all-trans RA, given orally on day 9 of gestation; embryos were taken 4, 12 or 24 hr after RA administration. The apoptotic pattern of RA-induced cell deaths was confirmed using the dUTP biotin nick-end labeling (TUNEL) method and transmission electron microscopy (TEM). HSP110 expression was detected using an immunohistochemical approach. The increase in the number of TUNEL-positive cells and HSP110-positive cells after all-trans RA administration was quantified in the first branchial arch using a computerized method. Twelve hours after RA administration, the increase in the number of HSP110-positive cells is greater than the increase in the number of TUNEL-positive cells. Twenty-four hours after RA administration, only TUNEL-positive cells remain strong in number. We suggest that HSP110 expression could represent a biochemical event of apoptotic cell death induced by RA, associated with early stages of the apoptotic process. In order to find out if HSP110 expression resulted from neosynthesis, we performed in situ hybridization, which demonstrated that the expression of HSP110 occurred at the level of mRNA.     

Assay of ribonucleotide reduction in nucleotide-permeable hamster cells

Ribonucleotide reduction was measured in Chinese hamster ovary cells made permeable to nucleotides by treatment with the detergent Tween-80. When compared to the respective ribonucleotide reductase activity in partially purified cell extracts, CDP and GDP reductase activities in permeabilized cells responded in a similar fashion to dithiothreitol, pH, MgCl2, FeCl3, substrate concentration and the presence of positive or negative allosteric effectors. At low protein concentrations both CDP and GDP reduction with whole cells increased linearly with cell number and was greater than the activity in corresponding cell extracts. Permeabilized cells were used to measure the level of CDP and GDP reductase in a hamster cell line resistant to the cytotoxic effects of hydroxyurea. The hydroxyurea-resistant cell line contained four to ten times more CDP and GDP reductase activity compared to parental or revertant cell lines. The permeabilized cell assay was also used to measure CDP and GDP reductase activities in Chinese hamster ovary cells synchronized by isoleucine starvation. CDP reductase activity was low in G1 arrested cells but increased 10-fold by 16 hours after the readdition of isoleucine to the growth medium. GDP reductase, which is present at much higher levels, is similarly induced after isoleucine addition, but only by 2-fold. The maximum activity of both CDP and GDP reductase occurred from 14 to 16 hours after isoleucine addition, which corresponded to the period of maximum DNA synthesis.     

氯化锂抑制A549细胞增殖及其对Polo-like激酶1转录活性的影响

利用糖原合成酶激酶3的抑制剂氯化锂作用于A549细胞,观察细胞形态与增殖的改变及其对Polo－like激酶1转录活性的影响.采用细胞计数检测细胞增殖,流式细胞术分析细胞周期变化;Western印迹检测磷酸化GSK3以及细胞周期相关蛋白p53、cyclin B1和Plk1的表达变化;RT-PCR检测Plk1 mRNA的表达;荧光素酶报告基因分析氯化锂对Plk1启动子活性的影响.结果显示,5 mmol/L氯化锂作用48 h后,A549细胞即发生明显的形态学改变,细胞增殖减慢并发生G2/M期阻滞;Plk1 mRNA和蛋白表达均升高,p53蛋白表达增强,而cyclin B1的蛋白表达无明显变化.氯化锂作用24 h后,可见pGL2-Plk1转染组中荧光素酶活性增高（与对照质粒相比,P<0.05）,48 h后更明显.以上结果表明, 氯化锂减慢A549细胞增殖,导致G2/M期阻滞,并能增强Plk1的启动子活性,促进Plk1的表达.     

Calculation of Steel's cell loss factor and of the parameters of cellular kinetics for a population with an exponential growth of the cell number and cell death at G0 phase with a probability equal to 1

The method of calculation of three cell kinetics parameters (the Steel's cell loss factor phi, the proliferative pool Pc, and the mean number m of the proliferating cells after mitotic division of one cell) was shown to be the same for the exponential growth state of cell number with cell death at the G0-phase, and for the exponential growth state with cell death occurring immediately after mitosis. The value of the mean number delta of non-proliferating cells that appeared after mitotic division of one cell is different for these two models of the exponential growth state with the equal values of the other three parameters (phi, Pc, and m). A method is proposed for calculating the parameter delta on the data of the percentage of labeled cells obtained in the experiments with continuous cultivation of cells in the nutrient medium containing 3H-thymidine. The kinetics of cell line HL-60 (the experimental data of Foa et al., 1982) can be described at the first approximation, by a model of the exponential growth state with the cell death at the G0-phase, with Pc = 0.80, phi = 0.24, m = 1.61, delta = 0.39, and the life time of the non-proliferating cells tQ = 24 hours.     

The Spacelab 3 simulation: basis for a model of growth plate response in microgravity in the rat

Data from Spacelab 3 (SL3) suggested that spaceflight significantly reduces the activity of the rat tibial growth plate. Animal processing after SL3 began twelve hours post-landing, so data reflect post-flight re-adaptation in addition to spaceflight effects. To determine if a twelve-hour period of weight bearing after seven days of unloading could affect the physes of spaceflown rats, the present study assessed the growth plate response to unloading with or without a reloading period. Rats were subjected to hind-limb suspension for seven days and then euthanized, with or without twelve hours of reloading. Activity of the growth plate was assessed by morphometric analysis. Rats suspended without reloading had reserve zone (RZ) height greater than controls, and shorter hypertrophy/calcification zone (HCZ) with fewer cells. The greater RZ was associated with a larger cell area, indicating a possible mitotic delay or secretion defect. Twelve hours of reloading decreased RZ height and cell number, and restored the number of cells in HCZ to control values, but the number of cells in the proliferative zone and height in HCZ were reduced. These results suggest the rebound response to preserve/restore skeletal function after a period of unloading involves an acceleration of growth associated with a decreased cell cycle time in PZ. Changes during the reloading period in this simulation support our hypothesis that the effects of spaceflight on SL3 growth plates were altered by changes that occurred post-landing. The similarities in response to unloading by suspension or during spaceflight are used to propose a model of growth plate response during spaceflight.     

Proliferative activity of Shvetz tumor following single and repeated irradiation]

Autoradiographic study of an experimentally-induced tumour following local irradiation in a dose of 600 rad showed no retardation of the cell cycle 6 to 12 hours after the irradiation. Marked reduction of the mitotic index (MI) and of the labeled nuclei index (LNI) was noted to the 96th hour after the irradiation. In repeated irradiation in a dose of 1200 rad at an interval of 18 hours there was revealed a marked reduction of the MI and of the LNI as a result of the block of the passage of cells from the G1-period into S. However, restoration of the cell proliferation uas noted by the 24th-48th hours. A high MI revealed at all the periods of investigation after repeated tumour irradiation at an interval of 24 hours was possibly caused by an increase in the time of mitosis proper, this also being confirmed by a significant accumulation of the number of late mitotic phases.     

DIURNAL VARIATION IN PROLIFERATIVE COMPARTMENTS AND THEIR RELATION TO CRYPTOGENIC CELLS IN THE MOUSE COLON

The depth of the crypts in mouse descending colon varied diurnally, between twenty-six cells at 24.00 hours and thirty-eight cells at 12.00 hours. Cell loss from the colon was greatest immediately before the maximum faeces production, at the beginning of the dark period. The labelling index of the colon also changed, from 9% at 20.00 hours to 16% at 12.00 hours. The greatest variation in labelling index occurred at the top of the zone of proliferative cells, between the ninth and eighteenth cell position up the crypt. In this region a synchronized cohort of about forty cells apparently entered S phase once a day. Although the length of the proliferative zone doubled at 12.00 hours, that of the non-proliferative zone remained fairly constant all day. The number of cryptogenic cells per crypt was estimated by comparing single and split-dose X-ray survival curves. This gave a mean value of two cryptogenic cells per crypt. Crypts rarely regenerated from the base after irradiation. The cryptogenic cells probably lay between cell positions Nos 9 and 18 up the crypt and probably did not function as stem cells in the normal crypt.     

人肝癌细胞表皮生长因子受体以及佛波酯对它的调度

Using radioligand binding assay, the presence of epidermal growth factor (EGF) receptors in cells of two human liver cancer cell lines, BEL-7402 and SMMC-7721, was demonstrated. The ligand binding data were analyzed by a computer program. The dissociation constants (KD) of the ligand-receptor binding complex at equilibrium for 7402 and 7721 cells were 1.2 nM and 0.8 nM respectively, and their number of EGF receptors per cell were 6.2 x 10(4) and 2.5 x 10(4) respectively. After the treatment of cells with phorbol 12-myristate 13-acetate (PMA), no change either in the affinity or in the number of EGF receptors was found in 7721 cells. However, in the case of 7402 cells, while the number of receptors, like 7721 cells, remained unchanged, the affinity of EGF receptors displayed a time dependent modulation after PMA treatment. It dropped within the first hour to a KD value of 3.0 nM and then gradually returned to the normal control value at 48 hours or even slightly higher than normal (0.95 nM) at 96 hours of treatment. The modulation or down-regulation of EGF receptors by PMA in 7402 cells was paralleled by the simultaneous inhibition of DNA synthesis in these cells as evidenced from their reduction of 3H-TdR uptake. It is not clear what is the basis for the differences found between 7402 cells and 7721 cells in their number of EGF receptors per cell and their responsiveness to PMA treatment. It might be related to their difference in autocrine secretion of alpha-transforming growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of the cell center to exposure to the calcium ionophore A-23187

Calcium ionophore A23187, taken at a concentration of 0.1 microgram/ml, quickly uncouples mitochondria in PE culture cells in medium 199. The cell ultrastructure undergoes reversible changes (especially that of mitochondria): maximum changes occur 2 hours after the start of the treatment; in 8 hours they become less pronounced. The adaptation of cells does not involve the ionophore inactivation in the medium. 10 micrograms/ml of A23187 induces gradual but irreversible alterations. Microtubules in PE cells are not destroyed when incubated in medium 199 containing 10 micrograms/ml of A23187 and 11 mM Ca2+. The addition of 10 micrograms/ml ionophore to the normal 199 medium (1.26 mM Ca2+) results in the formation of electron dense bodies in the cell center 30 minutes after the start of incubation. These bodies disappear in the course of a subsequent incubation. The number of cells with primary cilia decreases. The percentage of centrioles located perpendicularly to the substrate increases 30 minutes following treatment with 0.1 microgram/ml A23187 in medium 199. 2 hours after the start of treatment with 0.1 microgram/ml ionophore no such changes are detected; an electron dense halo appears around the centriolar cylinders. 8 hours after the start of treatment the structure of the cell center does not differ from the normal one.