Neoplasia in the connective tissue of the musselMytilus trossulus from polluted areas of Nakhodka Bay, Sea of Japan

A tumor was found for the first time in a musselMytilus trossulus from aheavily polluted area of Nakhodka Bay, Sea of Japan. Tumor cells were found in the connective tissue of different organs and also in gill vessels and hemal sinuses of the visceral mass. They were both attached and diffuse. The tumor was at an advanced stage, replacing the normal connective tissue cells, and formed nodes. The tumor cells were polymorphic, with a high nucleocytoplasmic ratio, and had a prominent nucleolus. The size of their nuclei was three to five times that of the nuclei of agranular hemocytes. The mitotic activity of the tumor cells was more than an order of magnitude higher than in the normal cells: The mean mitotic index was 1.4±0.5%, ranging from 0.97 to 2.3% in different organs. The mitotic indices in the connective tissue cells of three normal mussels were 0, 0, and 0.12%. A significant proportion (up to 78%) of the mitotic cells were at metaphase. The frequency of abnormal mitoses was 17%. Metaphases with displaced (often multiple) chromosomes constituted 71% of abnormal mitoses; anaphases, 8%; and tri- and tetrapolar mitoses, 11%. The tumor described is similar to diffuse sarcomatoid diseases of mussels from other geographical regions.     

Autoradiographic study of human scirrhous gastric carcinoma during cultivation in diffusion chambers]

The growth of the epithelial and connective tissue cells was noted in cultivation of human schirrous carcinoma of the stomach in diffuse chambers. In difference to the initial tissue of the tumour in vitro in which no incorporation of thymidine-H3 into the connective tissue cells was noted, these cells proved to be labeled under conditions of growth in the chambers. One hour after the administration of thymidine-H3 the percentage of cells with labeled nuclei averaged 25.1% this considerably exceeding the value of the label index determined under conditions of incubation in vitro of the initial tumour tissue (6.6%). Under conditions of cultivation there was revealed a rapidly proliferating subpopulation of cells with the mitotic cycle duration of 14.8 hours.     

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.     

Contribution on the physiological heart growth. (Experimental autoradiographic studies in mice)]

After injections of 3H thymidine or 3H proline, the physiological hearth growth in mice of the CBA strain belonging to various age groups was studied by means of autoradiography. The most important results are the following: The duration of the postnatal growth period is determined by the degree of maturity of the heart at the time of birth. It varies from species to species. 2. In the perinatal developmental phase the percentage of the 3H thymidine-labelled connective-tissue nuclei is higher than that of the muscle nuclei. In this period the connective supporting tissue is considerably strengthened. 3. During the postnatal developmental phase the DNA synthesis in the muscle nuclei aids the preparation of mitoses. After the postnatal duplication of cells the mitotic genes are repressed. The further growth is effected by the increase in weight of the individual fibres. 4. The process of growth is substantially determined by the intracardiac or intramyocardiac pressure and thus by the extension of the muscle fibre. Prior to birth the percentage of the labelled nuclei of muscle cells and connective tissue cells in the right ventricle was higher than in the left ventricular wall. In the postnatal period we observed a shift in the percentage of the labelled cells towards the left ventricular wall. The basis and the median section of the ventricular wall. The basis and the median section of the ventricular wall contain a higher percentage of labelled cells than does the apex cordis. During the first two weeks of live most of the DNA synthesising nuclei of muscle and connective tissue cells are localized in the two inner muscle shells. Later in life no clear distinctions can be demonstrated between the individual ventricular layers.     

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.     

Hunting the mechanisms of self-renewal of immortal cell populations by means of real-time imaging of living cells

The causes of the indefinite propagation of immortalized cell populations remain insufficiently understood, that hinders the research of such fundamental processes as ageing and cancer. In this study the interrelations between clonal proliferation and abnormalities of mitotic divisions in the immortalized cell line established from the mouse embryo were investigated with the aid of computerized microscopy of living cells. 3 mitoses with three daughter cells and 7 asymmetric mitoses which generated two daughter cells of conspicuously different sizes were registered among 71 mitotic divisions in the individual cell genealogy. Abnormal mitotic divisions either did not slow the proliferation in cell clones compared with progenies of cells that divided by means of normal mitoses or were followed by the acceleration of divisions in consecutive cell generations. These data suggest that abnormal mitotic divisions may contribute to the maintenance of the immortalized state of cell populations by means of generating chromosomal instability.     

Luteinizing hormone,progesterone and the morphological development of normal and superovulated corpora lutea in sheep

The development of granulosa-lutein cells was studied in 27 normal and 32 superovulated ewes between days 0-4(day 0 began with the preovulatory LH peak in normal animals and the HCG injection in superovulated ewes). The pattern of differentiation was similar in both groups. Following initial hormonal stimulation (0-12 hours after LH or HCG), granulosa cells were approximately 100 mu2 and contained small, pleomorphic nuclei with large amounts of clumped chromatin. Elongate cells lining the basement membrane possessed large, heterogeneous dense bodies, and a well-developed Golgi apparatus. Mitotic figures were observed up to 6 hours prior to ovulation. Sixteen to 20 hours following the LH surge or HCG injection, hypertrophy of granulosa cells was evident. Nuclei contained definitive nucleoli. Blood vessels in the theca interna were abundant and highly dilated. Ovulation occurred approximately 24 hours after the LH peak or HCG injection. Visible signs of luteinization were evident 6-12 hours after ovulation. A slight increase in serum progesterone levels was detected. The second post-ovulatory day was characterized by continuing hypertrophy of granulosa cells and extensive proliferation of smooth endoplasmic reticulum and mitochondria. Nuclei of granulosa cells were larger and possessed extremely large nucleoli. Numerous mitotic figures were apparent within the corpus luteum. Serum progesterone concentrations began increasing at 60-72 hours after hormone stimulation. By the end of the third post-ovulatory day, the corpus luteum consisted of large, pleomorphic, parenchymal cells, interspersed between capillaries and connective tissue elements. Only an occasional mitotic figure was apparent within the corpus luteum at 100 hours. Light microscopic autoradiography of 5, 10, and 15 day corpora lutea taken from ewes pulsed with 3H thymidine at specific times before and after ovulation revealed that granulosa cells did not undergo secondary mitoses following ovulation. In contrast, thecal, mesenchymal and endothelial cells did mitose on day 3.     

Embryo Sac Development in the Maize indeterminate gametophyte1 Mutant: Abnormal Nuclear Behavior and Defective Microtubule Organization

The indeterminate gametophyte1 mutation in maize has been known to disrupt development of the female gametophyte. Mutant embryo sacs have abnormal numbers and behavior of micropylar and central cell nuclei, which result in polyembryony and elevated ploidy levels in the endosperm of developing kernels. In this study, we confirm abnormal nuclear behavior and present novel findings. In contrast to the normal form, there is no obvious polarity in two-nucleate embryo sacs or in the micropylar cells of eight-nucleate embryo sacs. We show that the second and third mitoses are not fully synchronized and that additional mitoses can occur in all of the nuclei of the mutant embryo sac or in just the micropylar or central regions. After cellularization, individual micropylar cells can undergo mitosis. Abnormal microtubular behavior results in irregular positioning of the nuclei, asynchronous microtubular patterns in different pairs of nuclei, and abnormal phragmoplasts after the third mitotic division. These results indicate that in addition to acting primarily in controlling nuclear divisions, the indeterminate gametophyte1 gene acts secondarily in regulating microtubule behavior. This cytoskeletal activity most likely controls the polarization and nuclear migration underlying the formation and fate of the cells of the normal embryo sac.     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

Simultaneous fluorescence immunophenotyping and interphase cytogenetics: a contribution to the characterization of tumor cells.

In immunocytochemical studies, the phenotypic evaluation of tumor cells is often complicated by accompanying normal cells, representing the original tissue or infiltrating leukocytes. This holds particularly true for tissues with a great morphological and immunophenotypical variability, such as bone marrow. A method that identifies mitotic tumor cells by chromosomal aberrations and permits the subsequent immunophenotypical analysis was a first progress, demonstrated by Teerenhovi et al. However, the results are usually hampered by the low number of analyzable mitoses. We demonstrate here a method that simultaneously combines immunophenotyping and in situ hybridization with centromere-specific probes. Using our method, numerically aberrant tumor cells can be identified by interphase cytogenetics and subsequently analyzed immunophenotypically. Since all interphase cells can be analyzed, we are not limited by the number and banding quality of analyzable mitoses.     

TUMOR ANGIOGENESIS : Rapid Induction of Endothelial Mitoses Demonstrated by Autoradiography

Walker ascites tumor cells and an extract derived from such cells (tumor angiogenesis factor, TAF) were injected into the subcutaneous tissue of rats by using a dorsal air sac technique. At intervals thereafter, thymidine-³H was injected into the air sac and the tissues were examined by autoradiography and electron microscopy. Autoradiographs of 1µ thick Epon sections showed thymidine-³H labeling in endothelial cells of small vessels 1–3 mm from the site of implantation, as early as 6–8 hr after exposure to live tumor cells At this time interval endothelial cells appeared histologically normal. DNA synthesis by endothelium subsequently increased and within 48 hr new blood vessel formation was detected. The presence of thymidine-³H-labeled endothelial nuclei, endothelial mitoses, and regenerating-type endothelium was confirmed by electron microscopy. TAF also induced neovascularization and endothelial cell DNA synthesis after 48 hr. A similar response was not evoked in saline controls. Formic acid, which elicited a more intense inflammatory response, was associated with less endothelial labeling and neovascularization at the times studied. Pericytes and other connective tissue cells were also stimulated by live tumor cells and TAF. The mechanism of new blood vessel formation induced by tumors is still unknown but our findings argue against cytoplasmic contact or nonspecific inflammation as prerequisites for tumor angiogenesis.     

Temporal pattern of changes in mitotic frequency in the epidermis and other larval tissues of Bombyx mori

Temporal changes in mitotic frequency were examined in various tissues through late larval life of Bombyx mori. From the second larval ecdysis to the third and from the third larval ecdysis to the fourth, there was a definite temporal change of mitotic pattern in each tissue. In the epidermis as well as in the tracheal epithelium, mitoses began to appear about 1 day after an ecdysis, and showed a maximum 1 to 2 days after an ecdysis. In the fat body, mitoses were observed continuously through the instars, and the mitotic frequency showed a maximum state just before an ecdysis. In the abdominal muscle the frequency was highest at about the middle of the period between two successive ecdyses. Furthermore, epidermal mitoses coincided with the time when the density of epidermal nuclei per unit area decreased to a half. This suggests that epidermal mitoses may be initiated by some process related to the increase in cell size.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation.

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9-13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13-14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Dying and regeneration of human tumor cells after heterotransplantation to athymic mice

The histologic phenomena occurring immediately after heterotransplantation of two human colon adenocarcinomas to athymic mice have been studied. The tumors differed with respect to velocity of growth and passage age. Three phases were discernible in both cases. (1) During the first phase, most inoculated tumor cells died. (2) The second phase was characterized by removal of the necrotic tumor cells by immigrated inflammatory cells and by penetration of the connective tissue of host animals from peripheral into central areas of the implants. The first mitoses occurred within tumor cells in close proximity to these connective tissue septa. (3) During the third phase, signs of regeneration and proliferation of tumor cells resulted in the macroscopic enlargement of xenografts. Only in this phase, the typical histologic characteristics of the tumors were formed. These observations point to the host connective tissue invading into implants to be of great importance for the stimulation of tumor cell proliferation and, therefore, for the growth of xenografts. Thus, successful heterotransplantation is obviously based on mutual events between the transplanted tumor cells and host connective tissue.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9–13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13–14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Haymaker gene expression in malignant and normal gynecologic tissues.

We previously reported that cell lines established from human carcinomas and leukemias/lymphomas expressed high levels of an intracellular membrane-bound protein, Haymaker, whereas cell lines derived from non-malignant connective tissue cells and lymphoid cells expressed low levels of this gene product. To determine whether these findings reflect neoplastic transformation or, alternatively, tissue specificity, we examined by immunohistochemical and molecular methods the expression of Haymaker in gynecologic organs with and without tumor. A highly specific, affinity-purified rabbit polyclonal antibody against a 25-mer Haymaker peptide was used for immunohistochemical staining and morphometric analysis of 85 tissue specimens. Immunohistochemical studies demonstrate, for the first time, that Haymaker protein is highly expressed in epithelial cells of the endometrium of the normal uterus and to a somewhat lesser extent in the mucosa of the normal vagina and cervix, but is poorly expressed or absent in cells of the connective tissue and smooth muscle strata of these organs (p < 0.005). Significant differences in Haymaker expression, as assessed by immunohistochemistry, between malignant and normal gynecologic tissues were not observed (p = 0.27). The expression of Haymaker protein does not appear, therefore, to be a marker of malignant transformation of the epithelium of gynecologic organs but rather distinguishes both normal and malignant epithelial cells from normal connective tissue and smooth muscle cells.     

Circadian rhythm of the mitotic activity and of the number of nuclei synthesizing DNA in sarcoma 37 in white mice]

Circadian variations in the frequency of mitoses and the number of nuclei labed with thymidine-H3 in sarcoma-37 of mice were investigated. It was shown that the circadian rhythm of mitotic activity was composed of diurnal variations in the frequency of labeled and unlabeled mitoses. The G2-phase of mitotic cycle of the cells with labeled mitosis was approximately one hour. The G2-phase of the cells with unlabed mitosis lasted four hours and more. It is suggested that there are two cell populations in sarcoma-37.     

DNA synthesis in connective tissue elements of regenerating skin under the influence of thyrocalcitonin and hypoxia

The influence of TCT on the proliferation activity of the connective tissue elements of the regenerating skin in normal and lowered partial oxygen tension was studied by means of H3-thymidin autoradiography. Continuous saturation of the organism with exogenous TCT is characterized by an increase in the count of cells during the S-period of mitotic cycle, the DNA synthesis intensification, and a considerable decrease in the number of silver grains over the nuclei in the course of the 24-hour observation period; this can testify to the acceleration of the cell passage of mitotic cycle stages in normal and low partial oxygen tension in hypoxia.     

Effect of thyroxine on the diurnal rhythm of the mitotic activity and parameters of the life cycle of cells of the liver and esophagus]

The duration of the cellular cycle and the diurnal rhythm of the amount of mitosis were studied in young rats in normality and under the influence of thyroxin. The parenchymal and connective-tissue cells of the liver and cells of the liver and the cells of the oesophagus epithelium basal layer were studied. It was found that under the influence of thyroxin there occured a shortening of the periods of the cellular cycle and a 3--6 h shift to the left of the diurnal rhythm curve of the amount of mitoses. In thyroxinized animals the 21--95% increase of the amount of mitoses in the period of maximum values of the mitotic index during a day was observed as compared with control animals. A conclusion is made about the diurnal rhythm of sensitivity of G0-phase cells to the synchronizing factor, suggesting the decisive significance of the state of the cell population in the interaction of the tissue and hormone cells. The data obatained in the work show that the thyroid hormones regulate the cellular reproduction in the organism by stimulating the cells in the division cycle, synchronization of greater amount of cells by the moment of beginning of the mitotic cycle at a definite time of day and by shortening the period of the cell mitotic cycle.     

Host-supplied connective tissue as a guide for proliferating tumor cells in human tumor xenografts

The morphologic alterations of 7 human tumors of different origin and various histologic types, heterotransplanted to athymic nude mice, were investigated in the present study. Constant patterns of histologic and ultrastructural changes were observed in all 7 tumors. Following the initial dying of most inoculated tumor cells, host-supplied inflammatory cells invaded the xenografts and phagocytosed necrotic tumor cells. Fibroblasts which vivaciously produced collagenous material invaded the xenografts and built up solid strands of connective tissue which tightly contacted surviving tumor cells. These stands were used as guide-rails for ingrowing blood vessels. Immediately after their immigration, the first mitoses of tumor cells occurred in close proximity to capillary-conducting strands of connective tissue resulting in a revival of tumor cell proliferation near to the fibrous cords and a spreading of newly formed tumor cells along the strands of connective tissue. These results point to the host-supplied connective tissue as playing an important role for tumor proliferation and local tumor expansion.