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.     

Chromosome deletion [46,XX,del(20)(q11)] in agnogenic myeloid metaplasia

Summary A woman in the fourth year of agnogenic myeloid metaplasia was found to have partial deletion of the long arm of chromosome 20 [46,XX, del(20)(q11)] in mitoses of presumably immature myeloid cells from unstimulated cultures of peripheral blood and bone marrow. Cytogenetic studies of peripheral blood lymphocytes showed a normal female karyotype.     

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.     

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.     

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     

The mouse karyotype in somatic cells cultured in vitro

Summary An idiogram of the karyotype of the mouse somatic cells in culture was constructed by arranging the chromosomes according to decreasing order of length. The length of the individual chromosomes was then measured in 25 colchicin-blocked, hypotonic-treated metaphases from male C3H mice.It was shown that the 40 chromosomes of the diploid chromosome set of the mouse, although they appeared to be very similar to one another, can be subdivided into five distinct groups, each including chromosomes of similar length. The X chromosome belongs to the second group, the Y chromosome to the fifth group. It is thus possible to identify the sex also in somatic cells.In order to test the reliability of such a classification, a statistical analysis of the chromosome measurements in 25 mitosis of male C3H mice was carried out. The analysis has shown that the length differences between the chromosomes belonging to different groups are highly significant. A high variability among mitoses is however present.     

姐妹染色单体互换在鹌鹑胚胎细胞染色体间及染色体上的分布

SCE在鹌鹑胚胎细胞间的分布为Poisson分布;在染色体间的分布是非随机的,与染色体的相对长度成正相关（P<0.05）,但也不完全按各染色体的相对长度分布。着丝粒区的SCE相对很高,按染色体的相对长度分布。胚胎的不同性别对每个细胞的SCE平均值无显著影响,但对性染色体Z上的SCE是否有影响还不能做出肯定的结论。     

Requirement of mitoses for the reversal of X-inactivation in cell hybrids between murine embryonal carcinoma cells and normal female thymocytes

By means of a 5-bromodeoxyuridine (BrdU) incorporation and acridine orange fluorescence staining method we studied reactivation of the inactivated X chromosome (Xi) in newly formed cell hybrids between the near-diploid HPRT-deficient OTF9-63 murine embryonal carcinoma cell (ECC) with an XO sex chromosome constitution and the normal female mouse thymocyte. As reported earlier, most near-tetraploid hybrid cells were ECC in morphology and retained all chromosomes from both parents including three X chromosomes. Synchronization of the late replicating X chromosome in such hybrid cells, indicative of reactivation, was found for the first time on Day 3, and the frequency of reactivation was attained 90% on Day 5. Inhibition of cell cycle progression either by methylglyoxal bis(guanylhydrazone) dihydrochloride, an inhibitor of polyamine metabolism, or by isoleucine-deficient medium after cell fusion delayed reactivation of the Xi, which implied that the number of cell division cycles traversed by individual cells rather than the length of time after cell fusion is critical for the reactivation. Double-labeling experiments using [3H]thymidine and BrdU indicated that hybrid cells had undergone three or four mitoses before reactivation of the Xi. Most probably reactivation of the Xi is consequent to reversion of the thymocyte genome to an undifferentiated state under the influence of OTF9 genome. DNA demethylation or dilution of Xi-specific factors by mitoses may be involved in this process.     

MITOTIC ACTIVITY IN THE CAMBIAL ZONE OF PINUS STROBUS

Mitotic activity in the cambial zone of 20-year-old Pinus strobus L. trees was determined quantitatively, using mitotic counts from serial tangential sections of sample pieces. Counts from nine cores 1.6 mm in diameter from each sample piece were averaged and expressed as the number of mitoses per core with the sampling error. Estimation of the number of cambial zone cells per core permitted calculation of mitotic indices. In the fourth internode from the ground the first mitoses were observed on April 15 and the last on September 15. The number of mitoses per sample piece was comparable throughout each internode at any one sampling, but the number was higher in internodes within the crown than in those below it. Mitotic index was not appreciably higher within the crown, but it decreased from 3.7 in spring to 2.0 in fall. An internode in the crown sampled May 11–13 showed afternoon peaks in mitotic activity, but an internode below the crown showed no peaks, nor did other internodes sampled later in the season.     

Chromosome lesions in amniotic fluid cell cultures

Summary The frequencies of chromosome lesions were determined on 3537 mitoses in samples of varying sizes from cultures of 25 amniotic fluid specimens taken from patients at cytogenetic risk. The average percentage values of aberrant cells, including and excluding gaps, were 12.5 and 4.9, respectively. The corresponding values for fibroblasts and peripheral blood lymphocytes from normal adult donors, calculated under the same laboratory conditions, were 5.0 (including gaps) and 2.4 (excluding gaps) and 2.4 (including gaps) and 1.0 (excluding gaps), respectively. The hypothesis of a correlation between the increased incidence of chromosome lesions and the occurrence of abnormal karyotypes in amniotic fluid cell cultures is discussed.     

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.     

Further studies on pachytene pairing failure in maize

Summary Pairing failure in pachytene chromosomes was studied against a genetically constant background. Very significant negative correlations were found between total chromosome length per cell on the one hand and number of terminal pairing failures and their total extent (per cent length) on the other, and between number of terminal failures and their average extent (per cent length). No significant correlation was found, however, between total chromosome length per cell and the average extent (per cent length) of pairing failure. If the pairing failures are dissociations increasing in extent and number during the pachytene stage studied, the simplest reconciliation of the results requires that the average rate of their extension be roughly proportional to total chromosome length or that certain constants be related to each other as described. An alternate interpretation, that the pairing failures were present before pachytene and that chromosomes are shortening faster in cells containing them, is favored by the findings that heterogeneity is low between anthers in number of failures per cell and that no significant correlation was found between anthers in total chromosome length and number of pairing failures. The possibility is also discussed that the pairing failures are a complex combination of both initial synaptic failure and later dissociation.Distributions of chromosomes containing pairing failure at both ends and of those containing both terminal and intercalary pairing failures generally follow expectations of randomness.This work was supported by National Institute of Health Grant Number RA-6492 and by National Science Foundation Grant Number G 7068.     

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.     

Chromosome spatial order in human cells: evidence for early origin and faithful propagation

We have investigated the origin and nature of chromosome spatial order in human cells by analyzing and comparing chromosome distribution patterns of normal cells with cells showing specific chromosome numerical anomalies known to arise early in development. Results show that all chromosomes in normal diploid cells, triploid cells and in cells exhibiting nondisjunction trisomy 21 are incorporated into a single, radial array (rosette) throughout mitosis. Analysis of cells using fluorescence in situ hybridization, digital imaging and computer-assisted image analysis suggests that chromosomes within rosettes are segregated into tandemly linked “haploid sets” containing 23 chromosomes each. In cells exhibiting nondisjunction trisomy 21, the distribution of chromosome 21 homologs in rosettes was such that two of the three homologs were closely juxtaposed, a pattern consistent with our current understanding of the mechanism of chromosomal nondisjunction. Rosettes of cells derived from triploid individuals contained chromosomes segregated into three, tandemly linked haploid sets in which chromosome spatial order was preserved, but with chromosome positional order in one haploid set inverted with respect to the other two sets. The spatial separation of homologs in triploid cells was chromosome specific, providing evidence that chromosomes occupy preferred positions within the haploid sets. Since both triploidy and nondisjunction trisomy 21 are chromosome numerical anomalies that arise extremely early in development (e.g., during meiosis or during the first few mitoses), our results support the idea that normal and abnormal chromosome distribution patterns in mitotic human cells are established early in development, and are propagated faithfully by mitosis throughout development and into adult life. Furthermore, our observations suggest that segregation of chromosome homologs into two haploid sets in normal diploid cells is a remnant of fertilization and, in normal diploid cells, reflects segregation of maternal and paternal chromosomes. Received: 19 January 1998; in revised form: 28 May 1998 / Accepted: 30 June 1998     

Marker Chromosome Analysis of Tetraparental AKR↔CBA-T6 Mouse Chimaeras

Marker chromosome analysis of 18 tetraparental AKR↔CBA/H-T6 chimaeras revealed a great excess of AKR mitoses over CBA mitoses in direct preparations from lymphomyeloid tissues, corneal epithelium, intestinal epithelium and skin (Table 1). The degree of AKR dominance was strongly influenced by anatomical site (Tables 2 and 3). The testes of three out of four XY/XY males contained a marked excess of AKR germ cells and produced a parallel excess of functional AKR gametes (Table 4). Mitotic spreads in mitogen-stimulated cultures of tail blood were overwhelmingly of AKR type in 1972, but less so in 1973 (Table 5).
The coat phenotypes, and breeding results from known or presumptive XX/XX females, suggest that AKR and CBA cells were numerically balanced when melanoblasts and oocytes were formed during embryonic development, and therefore that the striking deviations from equality observed in mitotic populations of adult chimaeras arose later by differential proliferation or survival of AKR cells, or both.
The low frequency of lymphomas, compared with normal AKR mice, previously reported in these chimaeras cannot therefore be accounted for by insufficiency of AKR cells in the thymus or elsewhere in the lymphomyeloid tissues. One of three lymphomas studied was CBA type. This suggests that the high risk of lymphomatous transformation is not an autonomous property of AKR cells.     

Chromosome replication does not trigger cell division in E. coli

An essential part of the chromosome replication origin of E. coli K-12 and B/r was replaced by the plasmid pOU71. The average initiation mass of replication for pOU71 decreases with increasing temperature. The constructed strains were grown exponentially at different temperatures, and cell sizes and DNA content were measured by flow cytometry. The average DNA content increased with increasing temperature, but the cell size distribution was largely unaffected. Furthermore, cells in which DNA replication had not yet initiated (cells in the B period) became less abundant with increasing temperature. The increased DNA content could not be explained by an increase in the length of the C period. It is concluded that chromosome replication does not trigger cell division in E. coli, but that the chromosome replication and cell division cycles of E. coli run in parallel independently of each other.     

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.     

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.     

Studies of multipolar mitoses in euploid tissue cultures

Cultures of kidney epithelium and fibroblasts of 39 specimens of Microtus agrestis were investigated. In all 77 cultures multipolar mitoses were found. They were studied in living state and after pulse labelling with ³H-thymidine. The ploidy of the multipolar mitoses and of their daughter nuclei was determined by measuring the relative Feulgen-DNA content and by counting the predominantly constitutive heterochromatic sex chromosomes. Constitutive heterochromatin was demonstrated by late replication, retarded separation of the chromatids in anaphase, heteropycnosis and by the Giemsa technique of Arrighi and Hsu (1971). The latter stained also the spindle apparatus of mitoses.—In living cells, transformation of multipolar mitoses into bipolar mitoses was observed. The chromosomes of multipolar mitoses are separated into complete genomes; the daughter nuclei can be haploid, diploid, triploid or tetraploid. The chromosomes of haploid and triploid metaphases were studied with the Giemsa banding technique. The banding pattern shows an exact monosomy and trisomy, respectively, for each chromosome. Haploid nuclei are likely to be viable only in multinucleate cells, whereas triploid cells behave like diploid cells during the S period and the mitosis.Dedicated to Prof. Dr. K. Goerttler on the occasion of his 75th birthday.Supported by the Bundesministerium für Bildung und Wissenschaft of the Federal Republic of Germany.     

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.