Cell proliferation and chromosomal damage in human leukocytes: dicentries and premature chromosome condensations in first,second, and third mitoses after X-irradiation

Summary The frequencies of dicentric chromsomes and of partial premature chromosome contrnsation (PCC) in definitely first, second, or third mitoses after X-irradiation of human leukocytes, as determined with a bromodeoxyuridine (BUdR)/Giemsa labeling technique, were investigated. The results show that practically no PCCs occur in first mitoses after irradiation and culture start in contrast to second or third ones. The frequencies of dicentrics from first to second, as well as from second to third mitoses after irradiation decline by about 50% for each division step.     

Human cells in suspension 3

Summary After differential staining of sister chromatids, chromosome length distribution and average chromosome length were determined for human lymphoid cells in, respectively, the first, second, third, and fourth and later mitoses. A significant change in chromosome length distribution and a marked decrease in average chromosome length were found for cells in the fourth and later mitoses. This supports previous suggestions that the observed changes in these parameters in cell population during in vitro aging are due solely to an increase of cells in the fourth and later mitoses.     

The quantity of associated acrocentric chromosomes in human lymphocytes passing through 1st, 2d and 3d mitoses in culture

The reproductive ability of lymphocytes of peripheral blood with the usage of 5-bromine-deoxyuridine has been studied in 8 healthy children at the age of 5-6 years. Single second mitoses occurred in 48 hour cultures (6.5%), in 72 hour cultures the frequency of the first, second and third mitoses was equal, in 96 hour cultures the third mitoses dominated. Consequent divisions of lymphocytes were accompanied by a decrease in associative acrocentric chromosome, in average by 25%, within one mitotic cycle, while in mitoses of a given ordinal number the frequency of associations did not depend on the duration of cultivation. The fixation of the culture at the 48th hour of cultivation makes it possible to take into account the frequency of associations of acrocentric chromosomes without calculation of the ordinal number of mitosis because of an significant amount of second mitoses at this time, and of a sufficient value of the mitotic index (4.6 +/- 0.5%) necessary for cytogenetic analysis.     

The human leukocyte test system. V. DNA synthesis and mitoses in PHA-stimulated 3-day cultures.

In human leukocyte cultures st up with TC medium 199,DNA synthesis and mitotic indices were analysed by means of 3H-thymidine autoradiography and cell counting. DNA synthesis starts at around 28 hrs. The frequencies of labelled cells rise slowly and reach a maximum of around 24%. The first mitoses appear at around 38 hrs but up to 49 hrs only very few mitoses can be seen. After that time the mitotic indices rise and reach values of up to 11% cultivation in the presence of BudR for 72 hrs and staining with Hoechst 33258 stain revealed that first, second and third mitoses occur together in the cultures at this time. Irradiation of whole blood and cultivation for 72 hrs leads to mitoses containing dicentric and ring chromosomes with and without fragments, to interphases with micronuclei, to premature chromosome condensations (PCC) and to polyploid mitoses indicating that at this time first and further mitoses are present.     

Adaptive response in different mitotic cycles after irradiation

The frequency of cells with chromosome aberrations and the number of aberrations per cell have been studied by metaphase analysis in the nonirradiated progeny of irradiated human blood lymphocytes. DNA fragmentation (DNA double-stranded breaks) has been investigated by DNA comet assay. To study the adaptive response (AR), PHA-stimulated lymphocytes were irradiated by the adaptive dose (0.05 Gy) in 24 h and by challenge dose (1 Gy) in 48 h after stimulation. The first through fourth mitoses were identified by 5-bromodeoxyuridine. It was found that the frequency of chromosome aberrations and double-strand breaks were increased in all mitotic cycles after the challenge irradiation. In most individuals, the adaptive response is induced by adaptive and challenge irradiations in the first and the second mitotic cycles (48 and 72 h after stimulation, respectively); however, it is absent in the third and the fourth mitoses. In the first mitosis (1Gy in 48 h after stimulation), only chromatid aberrations are observed; chromosome aberrations were registered in subsequent mitoses. DNA comet assay showed that the adaptive response was obvious at 48–72 h, but not 96 h, after stimulation. It can be concluded that the nonirradiated progeny of irradiated lymphocytes have genomic instability. The adaptive response is manifested up to the third mitosis and is explained by the decreasing number of chromatid and chromosome aberrations and DNA fragmentation. We suppose that double-stranded DNA breaks may be damage signals for the induction of adaptive response.     

Satellite association frequency and number of nucleoli depend on cell cycle duration and NOR-activity

Summary In human lymphocyte cultures the frequencies of satellite associations in first, second, and third mitoses were investigated using the BUDR-method. A marked decrease of the association frequency with increasing numbers of cell cycles was found. The number of nucleoli seen in interphase is correlated with the satellite association frequency in the respective metaphase. Satellite association is positively correlated to Ag-staining intensity of the NORs. Individual differences in satellite associations are due to differences in NOR activity and in lymphocyte activation. BUDR diminishes somewhat the Ag-staining intensity of the NORs but has no effect on satellite association frequencies. The main reason for the decrease of satellite association frequency in second and third lymphocyte mitoses is presumably a certain dislocation of the original chromosome position during mitosis and a decreased possibility of association during the short interphases. The high association frequency in first mitosis resembles the chromosome position in the long interphase of G₀-lymphocytes.     

Postmeiotic mitoses without chromosome replication in a mutagen-sensitiveNeurospora mutant

In the recessive mutagen-sensitive mutantmus-8, no ascospores are formed. Ascospore delimiting membranes may be defective. Multiple postmeiotic mitoses occur, apparently without chromosome replication (at least at the first two mitoses). This results in many small nuclei, most of which contain only a few chromosomes or a single chromosome. These events resemble those in certain higher-plant mutants such as polymitotic inZea mays.     

The characteristics of the course of mitosis in polykaryons formed during cell fusion]

A new method of cell fusion is proposed utilizing treatment with 15% solution of DMSO in serum before and after PEG treatment. With such treatments in SPEV cell culture a higher rate of cell fusion was obtained than that with other known methods of cell fusion. In the first wave of mitoses (0.5-4 h) mainly asynchronous division of nuclei, premature chromosome condensation and formation of telophase-like nuclei were observed in polykaryons. In the period of the second wave (14-20 h), mitoses were mainly synchronous and completed with cytokinesis. Micronuclei were formed frequently as a result of such mitoses. After the first wave of mitoses the number of polykaryons with pycnotic chromosomes sharply increased, and after the second wave of mitoses the number of polykaryons with pycnotic nuclei increased. The results obtained allow to conclude that heterophasic condition of the fused cells is one of the causes of pathological mitosis of polykaryons and of their death.     

Premature chromosome condensation in a case of Fanconi's anemia.

Evidence is presented for the occurrence of premature chromosome condensation (PCC) from micronuclei even in the first in vitro mitoses in a case of Fanconi's anemia.     

Meiosis in a sterile male mouse with an isoYq marker chromosome

A male mouse with a metacentric Y chromosome of twice the normal size has been studied chromosomally in bone marrow mitoses, spermatogonial mitoses, and diakinesis-metaphase I primary spermatocytes. A low frequency of nondisjunction for this chromosome (2%) was noted in both bone marrow and spermatogonial mitoses. In spermatogonial mitoses, loss of the Y chromosome had occurred to the extent that 12% of spermatogonia were XO, resulting in 17% XO primary spermatocytes. Hardly any stages beyond the primary spermatocyte stage were encountered, which agrees with testis weights of approximately 30% of normal. Surface-spread pachytene spermatocytes yielded few cells that were analyzable for their total complement of synaptonemal complexes. The Y chromosome showed complete fold-back pairing and was located far away from the X chromosome. X and Y chromosomes were paired in 14.5% of the diakinesis-MI spermatocytes that contained a Y chromosome. The origin of this chromosome is discussed against the background of localization of the gene for the testis-determining factor on the short arm of the mouse Y chromosome.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The association pattern was studied in 1182 mitoses of 21 patients with trisomy 13 and in a control group. In addition, 173 trisomic mitoses were compared with the same number of diploid mitoses in a case of mosaicism.The number of mitoses with associations was no higher in the trisomic cells than in cells with normal karyotypes. Some differences were observed in the frequency of associations per cell and of the types of associations in the patient group and in the trisomic cells of the mosaic case. The number of associations in which more than two acrocentric chromosomes were involved was unexpectedly low in the cells with a supernumerary chromosome 13.The result are interpreted as suggesting the existence of a compensatory mechanism activated by the additional acrocentric chromosome.Parts of this work are included in the doctoral (MD) thesis of DM     

Sister chromatid exchange in lymphocytes in myelodysplastic syndrome

Sister chromatid exchange (SCE), percentage of first, second, third mitoses, blastic transformation index and mitotic index in patients with myelodysplastic syndrome (MDS) (3 with refractory anaemia, 2 with refractory anaemia with sideroblasts, 1 with refractory anaemia with excess of blasts, 4 with refractory anaemia with excess of blasts in transformation) and in 15 healthy volunteers were estimated. Three types of lymphocytes cultures were set up: first with phytohemaglutinin (PHA), second with PHA and bromodeoxyuridine (BRdU), third with BRdU. In healthy persons the SCE frequency was negatively correlated to proliferating rate index, but in MDS such correlation was not found. The lymphocytes cell cycle duration based on percentage of mitoses was longer in MDS patients than in controls. The results of our studies show the disturbances of lymphocytes during cell cycle division resulting in higher SCE frequency and lower proliferating rate compared to controls.     

Hansemann, Boveri, chromosomes and the gametogenesis-related theories of tumours

Theodor Boveri (1862-1915) is often credited with suggesting (in 1914) the first chromosomal theory of cancer, especially in terms of abnormal numbers of chromosomes arising in cells by multipolar mitoses in adult cells. However, multipolar mitoses in animal cells had been described as early as 1875, and Hansemann (1858-1920), in publications between 1890 and 1919, included this mechanism among various ways by which abnormal chromosome numbers might arise in cells and cause tumour formation. Both theories were conceived in a period when gametogenic ideas of tumour formation were current. Boveri based his theory on the observation that some cells in early sea urchin embryos having abnormal chromosome complements wander from their usual developmental paths. His observation may have been seen by other authors at the time as support for Cohnheim's "embryonic cell rest" theory of cancer. Hansemann's contribution is seen as both the original, and the more significant of the chromosomal theories of cancer.     

Pathology of the reproductive apparatus of cells under the action of remantadine and deitiforin

The antiviral effect of chemical agents remantadine and deutephorine on the reproductive apparatus of MDCK-cells was studied. It was established that the action of the chemical agents during 3-4 days induced enhancement in pathologically transformed mitoses. This enhancement was in direct proportion to the doses used and to the cultivation time. Dose dependent enhancement of the total pathological mitoses number was simultaneous with the redistribution of mitoses into groups: the relative number of pathologies connected with chromosome damage became lower and the number of degenerating mitoses grew. Computer regression analysis showed the linear dependence between the chemical agent dose and the number of mitoses in the metaphase, that gives an indirect evidence of statmokinetic action of the agents under study.     

Meiosis and spermatogenesis in G-Trisomic males

Summary In 3 adult males with trisomy-G, Down's syndrome, the chromosomes were studied in spermatogonial mitoses and in the first meiotic division. The findings were similar in all cases and are here presented together. Of 8 spermatogonial mitoses of good quality, 4 had 46 chromosomes and 4 had 47 chromosomes. At diakinesis-metaphase I, 21% of the cells had 1 G-bivalent and 1 trivalent, 38% had 2 bivalents and 1 G-univalent and in 41% 2 chromosomal elements composed of G-chromosomes were seen, but it was not possible to determine unequivocally whether 1 of them was a trivalent or not. From their morphology and from the spermatogonial chromosome counts it was tentatively concluded that at least some of them had only 2 G-bivalents. Premeiotic elimination of 1 G-group chromosome is a possible explanation of this phenomenon. The study of larger samples of spermatogonial mitoses should allow definitive conclusions to be made. — Different trivalent and univalent configurations were described. — Spermatogenesis was quantitatively assessed and found to be complete but of lesser magnitude than in a normal male. Spermatogenetic arrest was not noticed. As judged from histological sections of the testes, all 3 G-trisomic males would have to be considered fertile.     

Biological significance of liver cell polyploidy: an hypothesis

The liver cell polyploidy phenomenon, a characteristic of many species of mammals, is reviewed. The liver parenchyma of adult animals represents a mixed population of mononuclear and binuclear cells with different number of chromosome sets and, therefore DNA content per nucleus. The polyploid hepatocytes are formed during postnatal liver growth as a result of a change from normal mitoses to polyploidizing ones. Hence, the polyploidization of hepatocytes is regarded as an equivalent of cell multiplication.An hypothesis of the biological significance of liver cell polyploidy is based on the fact of a high level of spontaneous chromosome aberrations in mitotic hepatocytes. Ploidy increase is known to give resistance against different kinds of genome alteration. Polyploidization of the liver cells ensures protection against deleterious consequences of the aberrant genome formation resulting from aberrant mitoses.Some implications of the hypothesis are discussed: the reasons for species-specific differences of liver cell polyploidy; the mechanisms of hepatocyte radioresistance; the relation of polyploidy to liver cell aging. The prerequisite factors for unbalanced cell genome formation are adduced: DNA and chromosome damage as the first step in the process, stimulation of mitosis as the second one. The aberrant polyploid genome of hepatocytes is assumed to be the cytogenetic basis for some chronic liver diseases in man.     

Occurrence of premature chromosome condensation in mouse spermatogonia treated with busulfan

Busulfan induction of premature chromosome condensation (PCC) was examined in mouse spermatogonia. Adult (C3H X SWV) F1 male mice were injected intraperitoneally with busulfan at 10 or 30 mg/kg of body weight and killed 18-72 h later. Polyploid-like mitoses were frequent (ca. 1/4 of all spermatogonial mitoses examined) in both the untreated and treated groups. Most of these were considered to have been derived from normal spermatogonia. In busulfan-treated mice, polyploid-like mitoses with PCC were seen. The frequency of PCC-containing mitoses increased with increasing dose and exposure time. A possible interpretation for PCC induction in mouse spermatogonia is discussed.     

Chromosome Damage and Early Developmental Arrest Caused by the Rex Element of Drosophila Melanogaster

Rex (Ribosomal exchange) is a genetically identified repeated element within the ribosomal DNA (rDNA) of Drosophila melanogaster. Rex has a semidominant maternal effect that promotes exchange between and within rDNA arrays in the first few embryonic mitoses. Several of Rex's genetic properties suggest that its primary effect is rDNA-specific chromosome breakage that is resolved by recombination. We report here that rDNA crossovers are only a small, surviving minority of Rex-induced events. Cytology of embryos produced by Rex-homozygous females reveals obvious chromosome damage in at least a quarter of the embryos within the first three mitotic divisions. More than half of the embryos produced by Rex females die, and the developmental arrest is among the earliest reported for any maternal-effect lethal. The striking lethal phenotype suggests that embryos with early chromosome damage could be particularly fruitful subjects for analysis of the cell biology of early embryos.     

Occurrence and possible consequences of multipolar mitoses in primary cultures of adult rat hepatocytes

Proliferating primary cultures of adult rat hepatocytes are characterized by the occurrence of multipolar mitoses, and chromosome loss resulting in the formation of micronuclei at telophase. The percentage of multipolar mitotic figures was determined to be 12.76 ± 7.9%, 80% of which were tripolar. Multipolar mitotic stages showed a high incidence of chromosome loss, increasing from meta- (61.7 ± 16.6%) to telophase (72.1 ± 19.3%). Regular bipolar mitotic figures on the other hand also showed chromosome loss, however, to a lesser degree and decreasing from meta- (49.5 ± 10.4%) to telophase (34.9 ± 7.9%). The incidence of chromosome loss even in regular mitotic figures is very high compared to other cells and appears to depend on another special feature of hepatocytes: they remain flat and well attached during mitosis, so that shearing forces could be responsible for the separation of chromosomes from the mitotic spindle. Additionally this morphology creates a situation allowing for a maximal interaction of mitotic spindles of binucleated cells, leading to the high rate of multipolar mitoses observed. Both multipolar mitoses and chromosome loss could also explain the consecutive detachment of hepatocytes reported for proliferating primary cultures, since the aneuploid daughter cells generated can be expected to be nonviable in most cases and eventually detach. © 1993 Wiley-Liss, Inc.