Transient inhibition of L929 cell mitosis and locomotion by argon ion laser irradiation

Summary Argon ion laser irradiation of L929 cells transiently inhibits both entry into and passage through mitosis without affecting clonogenic survival. Anaphase mitotic figures virtually disappear from irradiated cell monolayers although prophase + metaphase mitotic figures can still be identified. The total number of mitotic figures does not change significantly and time-lapse video recording shows that cells do not enter mitosis following irradiation. This effect is dependent on light dose within the 900–2700 J/cm² range and persists for 10–48 h depending on the initial light exposure. Inhibition of cell locomotion and subsequent recovery were observed to occur over a similar time course. The possible contribution of these phenomena must be considered whenever biological systems are exposed to argon ion laser irradiation.     

Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures]

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.     

Dynamic accumulation and redistribution of methylmercury in the lens of developing zebrafish embryos and larvae

Neurotoxic methylmercury compounds are widespread in the environment and human exposure worries many communities worldwide. Despite numerous studies addressing methylmercury toxicity, the detailed mechanisms underlying its transport and accumulation, especially during early developmental stages, remain unclear. Zebrafish larvae are increasingly used as a model system for studies of vertebrate development and toxicology. Previously, we have identified the lens epithelium as the primary site for cellular mercury accumulation in developing zebrafish larvae (Korbas et al. in Proc Natl Acad Sci USA 105:12108–12112, 2008). Here we present a study on the dynamics of methylmercury accumulation and redistribution in the lens following embryonic and larval exposure to methylmercury l-cysteineate using synchrotron X-ray fluorescence imaging. We observed highly specific accumulation of mercury in the lens that continues well after removal of fish from treatment solutions, thus significantly increasing the post-exposure loading of mercury in the lens. The results indicate that mercury is redistributed from the original target tissue to the eye lens, identifying the developing lens as a major sink for methylmercury in early embryonic and larval stages.     

Distribution of mitoses within a lobule of the rat liver in recovery after various types of injuries]

The distribution of mitotic activity inside the rat's liver lobe was found to depend on the time that elapsed after the injury. At the early stages, the maximal quantity of mitotic figures in hepatocytes was observed along the periphery of the lobe, being less in the middle parts. At the later stages, mitotic figures were noted around the central veins. With the prolonged injury of the liver, the maximal amount of dividing hepatic cells was localized in the middle sections of the liver.     

A new action for topoisomerase inhibitors.

Topoisomerases are known to aid DNA replication by breaking and resealing supercoiled DNA. Consequently, cells exposed to topoisomerase inhibitors before or during the S (DNA synthetic) phase of the cell cycle undergo abnormal DNA replication and become irreversibly blocked in the G2 (pre-mitosis) phase. We report that following a 4-h exposure to topoisomerase II inhibitors, murine erythroleukemic cells (MELC) do not form mitotic figures but exhibit a time-dependent progression into G2 (4N DNA) and greater than G2 (up to 8N DNA) stages of the cell cycle. Following exposure to the topoisomerase I inhibitor camptothecin, recovering MELC also exhibit greater than G2 polyploidy, but to a considerably lesser degree: mitotic figures are present and a subpopulation of cells resumes cycling. However, both topo I and topo II inhibitors induce maximal percentages of greater than G2 cells when synchronized MELC are in the G2/M phase at the time of exposure. This suggests that, in addition to their S-phase action, topoisomerase inhibitors can interfere with chromosome condensation during G2 and, in so doing, induce polyploidy.     

A selective stain for mitotic figures, particularly in the developing brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

A Selective Stain for Mitotic Figures, Particularly in the Developing Brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

Proliferation patterns in the canine endometrium during the estrous cycle

The proliferative activity in the endometrium of 58 bitches in different stages of the estrous cycle was assessed by immunohistochemical detection of the Ki-67 proliferation associated nuclear antigen and by counting mitotic figures. The Ki-67 labelling index and the mitotic index were determined in the surface epithelium, the stroma, the crypts and the basal glands by calculating the percentage of Ki-67 positive cells and mitotic figures, respectively, on a total of 500 cells of each category. Endometrial vascular proliferation was also verified by counting the number of Ki-67 positive cells on a total of 100 endothelial cells. The present study showed two proliferation peaks involving different cell groups. In the surface epithelium, the stroma, the blood vessels and the crypts, the highest labelling and mitotic indexes were noticed during proestrus, whereas for the basal glands these indexes significantly increased (P < 0.05) during estrus compared to late metestrus and anestrus. Furthermore, a slightly positive correlation (P < 0.05) was found between the labelling index in the basal glands and the serum progesterone levels, whereas the labelling indexes in the other cell groups were positively correlated with the estradiol-17 beta levels, although not always significantly. These findings suggest that regulation of the proliferation in the surface epithelium, the stroma, the blood vessels and the crypts is different from the proliferation in the basal glands.     

Spatial and temporal patterns of deoxyribonucleic acid synthesis and mitosis in the endometrial stroma during decidualization in the pseudopregnant rat

A quantitative study was made of the spatial patterns of stromal cell mitosis and DNA synthesis in the endometrium of the pseudopregnant rat before and during decidualization. A colchicine block was used for mitotic counts, and DNA synthesis was studied by [3H] thymidine autoradiography. Observations were also made on the subsequent fates of [3H] thymidine-labeled stromal cells. Before the onset of decidualization, on Days 3 and 4 (vaginal cornification = Day 0), mitosis was largely confined to the subepithelial stroma along the sides and around the antimesometrial pole of the lumen. [3H] thymidine labeling and stromal mitosis following a decidualizing stimulus at noon on Day 4 of pseudopregnancy were first seen close to the uterine lumen, with subsequent spread to deeper layers of the endometrium. At noon on Day 5, mitotic figures were numerous on all sides of the lumen and at all depths in the endometrium. At later stages, mitosis and the development of polyploidy continued in the decidual tissue, but little DNA synthesis or mitosis occurred in the basal zone of the stroma adjacent to the myometrium. In this zone, many cells in animals given [3H] thymidine 18 to 24 h after induction of decidualization remained heavily labeled throughout the growth and regression of deciduomata. Labeled cells derived from the basal zone and outer edge of the decidual capsule were present in the stroma of the regenerated endometrium following the regression of deciduomata. It was concluded that although cells at all depths in the endometrial stroma undergo DNA synthesis and mitosis in the early stages of response to a decidualizing stimulus, their subsequent behavior and fate depend upon their position in the endometrium.     

Kinetochores and chromatin diminution in early embryos of Parascaris univalens

In Parascaris the mitotic chromosomes of gonial germline cells are holocentric and possess a continuous kinetochore along their entire length. By contrast, in meiotic cells, the centromeric activity is restricted to the heterochromatic tips where direct insertion of spindle microtubules into chromatin without any kinetochore plate is seen. In the presomatic cells of early embryos, which undergo heterochromatin elimination, only euchromatin shows kinetic activity. After developing a technique to separate the very resistant egg shell from the embryos, we studied the cell divisions during early embryogenesis by immunochemical and EM approaches. The results reported here show that in presomatic cells microtubules bind only the euchromatin where a continuous kinetochore plate is present. We also report observations suggesting that the binding of the long kinetochores to the mitotic spindle initiates to a limited number of sites and extends along the entire length, during chromosome condensation. The existence of different centromere stages in different cell types, rends Parascaris chromosomes a very good model to study centromere organization.     

Scoring mitotic activity in longitudinal sections of crypts of the small intestine

Various counts have been made of the number of mitotic figures in whole crypts and sections of crypts of the small intestine of the mouse. Samples were analysed from animals killed at different times of the day and at different times after administration of vincristine. Measurements have been made of the size of mitotic and interphase nuclei and of the radial position of mitotic figures. The correction factor, f, which is required to take into account the enhancement of mitotic counts in sections as a consequence of their centripetal position has been investigated. The results indicate the following: (1) transverse sections of the crypt differ from longitudinal sections if they involve cutting the intestine before fixation which may result in a relaxation of the crypt and its widening by 25%; (2) columnar cell nuclei have a shape that resembles a sphere flattened so that the average diameter is 20% greater in crypt transverse sections; (3) mitotic nuclei tend to be about half-way between the crypt edge and the central axis of the crypt; (4) between about four and seven times more mitotic figures have their mitotic axis parallel to the long axis of the crypt; (5) about one-third of all mitotic figures in a crypt are seen in a longitudinal section of the crypt. If this is related to the number of cells in the crypt as a whole and in a section, a correction factor fD for the mitotic index of 0.59 is obtained; (6) the correction factor fT derived from the shape and position of the mitotic figures measured in 3 microns longitudinal sections is 0.53; (7) relating cell cycle and mitotic accumulation data using a computer-based model of the crypt also permits a correction factor fmod to be estimated. This gives a value of 0.66. When sectioned material is used to calculate a mitotic index the most appropriate correction factor is fD; for mouse small intestine it is 0.59.     

Quantitative study of the effects of maternal hyperthermia on cell death and proliferation in the guinea pig brain on day 21 of pregnancy

On embryonic day 21, pregnant guinea pigs were exposed to a 44 degrees C environment for 1 hour. As a result, all brain ventricular zone cells in M phase of the mitotic cycle when heat-shock occurred became immediately pyknotic and all cell division was stopped for 4-8 hr. The pyknotic cells were removed at a definable rate until mitosis resumed, after which removal occurred in an apparently random manner. Long delays in the return to mitosis were related to increased destruction of S-phase cells deep within the ventricular zone and largely confined to the alar lamina. Upon recovery, a rostrocaudal delay in mitosis was apparent, and the number of mitotic figures was increased compared with control numbers for 1 hr, after which they returned to control numbers. It was evident that up to 40% of the cells within the ventricular zone were destroyed following brief maternal heat stress.     

Characteristics of trophoblast cell reproduction in the connective zone of the rat placenta. II. Determination of the degree of ploidy of mitotic shapes

Morphological and cytophotometric studies have been made on polyploidization of placenta connective zone cells. Measurement of the DNA content in mitotic figures show that within a period of development ranging from day 13 to day 14 the bulk of mitoses (up to 25%) become tetraploid and octaploid. This may suggest that polyploidization of placenta connective zone cells proceeds via incomplete polyploidizing mitoses. Among tetraploid and octaploid mitotic figures, there are those corresponding to all the mitotic stages, from prophase to telophase. Consequently, mitosis in tetraploid and octaploid cells can reach telophase. In such cases polyploidization is likely to follow the acytokinetic mitotic pattern. A question of a certain maximum level of polyploidy that may be reached by cells due to the incomplete mitosis is discussed.     

Scoring Mitotic Activity In Longitudinal Sections of Crypts of the Small Intestine

Abstract. Various counts have been made of the number of mitotic figures in whole crypts and sections of crypts of the small intestine of the mouse. Samples were analysed from animals killed at different times of the day and at different times after administration of vincristine. Measurements have been made of the size of mitotic and interphase nuclei and of the radial position of mitotic figures. the correction factor, f, which is required to take into account the enhancement of mitotic counts in sections as a consequence of their centripetal position has been investigated. the results indicate the following: (1) transverse sections of the crypt differ from longitudinal sections if they involve cutting the intestime before fixation which may result in a relaxation of the crypt and its widening by 25%; (2) columnar cell nuclei have a shape that resembles a sphere flattened so that the average diameter is 20% greater in crypt transverse sections; (3) mitotic nuclei tend to be about half-way between the crypt edge and the central axis of the crypt; (4) between about four and seven times more mitotic figures have their mitotic axis parallel to the long axis of the crypt; (5) about one-third of all mitotic figures in a crypt are seen in a longitudinal section of the crypt. If this is related to the number of cells in the crypt as a whole and in a section, a correction factor f_d for the mitotic index of 0.59 is obtained; (6) the correction factor f_T derived from the shape and position of the mitotic figures measured in 3 μm longitudinal sections is 0.53; (7) relating cell cycle and mitotic accumulation data using a computer-based model of the crypt also permits a correction factor f_mod to be estimated. This gives a value of 0.66. When sectioned material is used to calculate a mitotic index the most appropriate correction factor is f_D; for mouse small intestine it is 0.59.     

Some Characteristics of DNA Synthesis and the Mitotic Cycle in Ehrlich Ascites Tumor Cells

In vivo studies of Ehrlich ascites tumor cells during the first 5 days of growth in peritoneal cavities of mice consisted of the following: 1. Determination of growth curves by direct enumeration of cells. 2. Estimation of the duration of each phase of the mitotic cycle based on incidence of cells in different phases. 3. Radioautographic studies to determine the proportion of cells in different phases of the mitotic cycle that incorporate tritiated thymidine during a single brief exposure to this precursor of DNA. 4. Estimation of the rate of incorporation of tritiated thymidine at different times during the period of DNA synthesis by comparison of mean grain counts over nuclei in radioautographs at different times following exposure to tritiated thymidine. The assumptions underlying these experiments and our observations concerning the duration of the period of DNA synthesis and its relation to the mitotic cycle are discussed. It is concluded that DNA synthesis is continuous, occupying a period of 8.5 hours during the interphase and that the average rate of synthesis is approximately constant.     

Influence of gonadotropin (FSH + LH) and thyrotropin (TSH) on the multiplication of Chinese hamster ovary (CHO) cells. Impact of the age of the culture.

CHO cells repeatedly treated with gonadotropin showed peak division rates after their third exposure and a decrease in the mitotic rate after their fourth exposure. Thyrotropin induced a considerable decrease in the mitotic rate following the first exposure, a significant increase after the second and a further decrease following the third and fourth exposures. The pattern did not differ between the two hormones when the cells were exposed further. The age (density of the cell cultures) had an appreciable influence on hormone-provoked changes in the mitotic rate, this differing only in intensity and never in the response following the initial re-exposure.     

The effects of vinblastine in assessment of the influence of age on proliferative activity of murine palate and footpad epithelium

Abstract
Data concerning changes in the rate of cell proliferation of stratified epithelia with increasing age are conflicting. In the present study young (3-month-old) and old (22-month-old) C57B1/6NNia male mice were injected intraperitoneally with 2, 3, 4 or 8 mg vinblastine sulfate/kg body weight and killed after 1.5, 3, 4.5 or 6 h. The number of arrested metaphase figures per 1000 basal cells was counted in histological sections. Data were analysed using a multivariate analysis of variance. There was a significant difference between the accumulation of mitotic figures in footpad epidermis and palate epithelium and both tissues contained an increased number of mitotic figures with increasing periods of accumulation at all dose levels. In the footpad epidermis neither the age of the animal nor the dose of vinblastine had a significant effect on the number of mitotic figures. In contrast, for palate epithelium the accumulation of mitotic figures was significantly less in the old mice compared with the young mice and at a dose of vinblastine of 2mg/kg compared with the higher doses. There was a statistically signifycant interaction between the dose of vinblastine and its period of action. It was concluded that the different tissues manifest a differential sensitivity to vinblastine and that only palate epithelium showed a significant reduction in proliferative activity with age.     

Studies on the Growth of Spinach Leaves (Spinacea oleracea)

The growth of spinach leaves has been studied from approximately1 cm long to full size. Over-all growth was measured in termsof area and total number of cells. The differential growth ofleaves was measured by the changes in the shape of squares drawnon the leaf surface. Growth differentials in terms of numbersof cells and number displaying mitotic figures were measuredin leaf discs taken from different positions within leaves. It was found that cell division in spinach leaves continueduntil the leaves reach from one-third to one-half full size.Cell division within the lamina of the leaves was not uniformbut ceased at an early stage of development in the leaf tipregion and continued for an extended period at the base.     

The characterization of high-resolution G-banded chromosomes of man

The detailed characterization of G-banding patterns of high resolution human chromosomes has been possible with the utilization of a refined cell synchronization technique which routinely yields a large number of excellent quality cells in late prophase, prometaphase, early metaphase, and mid-metaphase. These mitotic cells exhibit up to a 400% increase in the number of bands previously visualized by standard methods. From studies of the banding patterns, it has become evident that the G-positive and, to some extent, the G-negative bands of mid-metaphase results from a coalescence of finer subbands of earlier stages and that each band and its corresponding subbands maintain a constant location throughout the process of chromosome condensation. A precise schematic representation of the number, position, height and staining intensity of bands is presented for the five largest chromosomes of the complement at the four mitotic stages.     

Self-replication of somatostatin cells in the antral mucosa of rodents

The possibility that antral somatostatin cells have a self-replicating activity has been studied in three species of rodents: mice, rats and guinea-pigs, after a flash tritiated thymidine injection. The immunocytochemical staining of somatostatin cells, using specific antiserum, was combined with radioautographic procedures. The labelling index for somatostatin cells--and for gastrin cells identified on serial sections--was established after counting a large number of cells at the optical microscope level, on parallel tissue strips removed throughout the entire antrum. A significant percentage of the somatostatin cell population synthesized DNA. Values were similar for the three species of rodents ranging from 0.8 to 1.1%, that is slightly higher than the percentage of labelled gastrin cells, which was 0.67-0.7%. After a 36-hr continuous infusion of radioactive precursor in one rat, the labelling index observed remained low; 2.33% for somatostatin cells and 1.68% for gastrin cells. Colchicine injection in mice allowed the observation of mitotic figures in well differentiated somatostatin cells. Four hours after that injection, the mitotic index was estimated roughly at 0.3%. Thus, evidence has been presented that in rodents a fraction of the antral somatostatin cell population is capable of dividing, similar to the situation in gastrin cells.