Entry into mitosis in vertebrate somatic cells is guarded by a chromosome damage checkpoint that reverses the cell cycle when triggered during early but not late prophase.

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.

X-ray-induced synaptonemal complex damage during meiotic prophase in female fetuses of Rattus norvegicus.

The different types of damage (synaptic anomalies, chromosome reorganizations and nucleolar fragmentation) observed in oocytes from female rat fetuses irradiated at 14, 16 and 18 days of gestation (d.g.), respectively, indicates the existence of a special sensitivity at the different stages of prophase to X-rays. At the highest dose (5 Gy), we observed a decrease in some of the synaptonemal complex indicators of chromosomal damage, probably reflecting a selection against cells with the highest degree of chromosome anomalies.

Chromosome damage and non-disjunction measured at the first cleavage division in normal and chromosomally mutant female mice irradiated at the diakinesis stage of female meiosis.

We have irradiated primary murine oocytes at the diakinesis stage of the first meiotic division with 0.6 Gy X-rays. Fertilized oocytes were cultured overnight to arrest the first cleavage division and display pronuclear chromosomes. All preparations were preferentially stained for centric constitutive heterochromatin and analyzed for structural and numerical radiation effects. Females of 3 different karyotypes were irradiated (all on a Swiss random-bred genetic background): +/+ (221 female pronuclei analyzed), Rb(11.13)4Bnr T(1;13)70H/Rb(1.13)4Bnr T(1;13)70H with 11.13(1) and 1(13) large and small marker bivalents (RbT/RbT, 242 zygotes analyzed) and the same karyotype but with a 1(13)H;1(13) Wa heteromorphic bivalent (RbT/RbT*, 126 zygotes analyzed). Hyperploid chromosome counts were encountered with frequencies of 11.8% (+/+), 11.9% (RbT/RbT) and 16.6% (RbT/RbT*). In this order of karyotypes, the frequencies of dicentrics per zygote were 0.07, 0.16 and 0.11 and the frequencies of fragments 0.13, 0.18 and 0.31. In about half of the supernumerary chromosome spreads, a dicentric chromosome was included. The long marker bivalent 11.13(1) had a non-disjunction frequency of 2.5 times its control value, partially because it was involved in dicentric formation as well. For the RbT/RbT karyotype, the spontaneous maternal non-disjunction level was 5.4%. For the RbT/RbT* karyotype, it can be assumed to be the same or slightly higher because of the 1(13)H;1(13) Wa heteromorphic bivalent. This increased intrinsic sensitivity for non-disjunction was not expressed as an increased sensitivity for aneuploidy after irradiation. This fact and the numerical association between hyperploidy and dicentric formation, both for normal bivalents and for the 11.13(1) marker bivalent, lead us to suppose that in the female mouse, irradiation-caused aneuploidy is effectuated via chromatid exchange. The data presented do not rule out the existence of another mechanism.

Efficient repair of DNA damage induced by heavy ion particles in meiotic prophase I nuclei of Caenorhabditis elegans.

The effects of heavy ion particle irradiation on meiosis and reproductive development in the nematode Caenorhabditis elegans were studied. Meiotic pachytene nuclei are significantly resistant to particle irradiation by the heavy ions carbon and argon, as well as to X-rays, but not UV, whereas diplotene to diakinesis stage oocytes and early embryonic cells are not. Chromosomal abnormalities appear in mitotic cells and in maturing oocytes irradiated with heavy ion particles during the diplotene to the early diakinesis stages, but not in oocytes irradiated during the pachytene stage. The pachytene nuclei of ced-3 mutants, which are defective in apoptosis, are similarly resistant to ionizing radiation, but pachytene nuclei depleted for Ce-atl-1 (ataxia-telangiectasia like 1) or Ce-rdh-1/rad-51 are more sensitive. Pachytene nuclei thus appear to effectively repair heavy ion-induced DNA damage by the meiotic homologous recombination system.

The influence of essential phospholipids (ESSENTIALE) on liver regeneration in gamma irradiated rats.

Gamma irradiation with a dose of 5.7 Gy within 30 min before partial hepatectomy (PHE) caused latent damage in the intact rat liver. This was expressed in the course of proliferation induced in the liver remnant by inhibition of the regenerative process, which was indicated by a decreased mitotic index and cellularity, an increased ratio of metaphases/prophases and a high chromosomal aberration frequency. The preparation of essential phospholipids (ESSENTIALE) that was injected in a dose of 360 mg/kg (i.p.) either 24 h before irradiation or 30 min after irradiation or repeatedly before and after irradiation, markedly stimulated the process of liver regeneration after PHE in both nonirradiated and irradiated rats. It moderated all the alterations induced by irradiation, especially changes in cellularity. The most effective was the repeated administration of ESSENTIALE whereas its single administration before irradiation was more effective than that after irradiation. Our results suggest that ESSENTIALE has not only a stabilizing effect on cell membranes, but also mitigates damage of genetic material induced by irradiation.

[Prolongation of occult damage to the liver after cessation of chronic gamma irradiation]. / Prodolzhitel'nost' skrytogo povrezhdeniia pecheni posle okonchaniia khronicheskogo gamma-oblucheniia.

A study was made of the effect of continuous gamma-irradiation on changes in the mitotic index (MI), the ratio of the number of metaphases to that of prophases (M/P), and the frequency of chromosome aberrations at the post-metaphase in the regeneration rat liver. The data obtained indicate that the injury to the intact liver of exposed rats persists throughout the entire period of observation which is displayed by a relative stability of M/P ratio and the frequency of chromosome aberrations. On the other hand, the MI changes indicate the trend toward the repair of the injury.

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation.

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK1. When the laser power delivered to sample surface was 90-190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360-420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50-70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.

Abortive conjugation induced by UV-B irradiation at meiotic prophase in Tetrahymena thermophila.

Conjugating Tetrahymena were irradiated by ultraviolet-B (UV-B) at various stages of conjugation. When the conjugants were exposed to the UV-B at late meiotic prophase (the stage from pachytene to diplotene), abortive conjugation was induced a high frequencies. After completing meiosis, a significant number of the conjugants showed marked anomalies, i.e., failure of nuclear selection after meiosis, and abortion of the subsequent conjugation process such as a postmeiotic division to form gametic nuclei, nuclear exchange, synkaryon formation, and postzygotic development. The conjugating pairs retained the parental macronucleus and separated earlier as compared with a control. The resultant exconjugants degenerated meiotic products and became amicronucleates. These observations strongly suggest the presence of a UV-sensitive molecule that is expressed specifically at the meiotic prophase and that directs the subsequent development after meiosis.

Distribution and relation of radiation induced chromosomal aberrations in human lymphocytes as seen in the first and second cell cycles.

Different types of chromosomal damage in the G(o) phase of human lymphocytes have been studied in the first (M1) and second cell lymphocytes (M2). Large populations of cells containing one dicentric chromosome and one acentric (DIC+) were observed in the second cycle. This indicates that these dicentric and acentric fragments have a normal distribution in M2 or, arose through loss of one acentric fragment (AC) from cells containing one dicentric and two acentric fragments (DIC++) in M1. Similarly, a large number of metaphases in M2 have unpaired differentiated ACs. Our data indicates that aberrant metaphases containing single AC or a DIC+ may undergo normal cell division and that these cells may not represent cells in the first cycle only, as was suggested by earlier workers. For radiation experiments to G(o), a desirable time to harvest human lymphocyte cultures appears to be 72 hours and screening of M1 and M2 cells by sister chromatid differentiation.

Meiotic delay of mouse spermatocytes carrying x-ray-induced translocations.

The kinetics of spermatocyte progression through meiotic prophase in cells with or without induced translocations were studied in mice that had been exposed to x-rays. Pulse-labeling experiments using 3H-thymidine, followed by autoradiographic analysis, indicated that at higher x-ray doses (6 and 7 Gy), translocation-carrying cells tend to spend more time in meiotic prophase than do normal cells. At 2 Gy, no such delay seemed to be present. The observed delay may explain the reduction in transmission of translocations to the next generation reported by others.

Effect of 1 Gy X-rays in G1 phase on activation of cell cycle checkpoints in human lymphocytes: role played by chromosomal aberrations.

The effect of X-irradiation (1 Gy) during G1 on the transition from G1 to S and on the length of G2 over cell cycles subsequent to irradiation was studied in human lymphocytes from six different donors. After irradiation a delay was observed in the onset of S phase, as was an extension of the G2 phase lasting throughout the three to four subsequent cell divisions. The extension of G2 and of the cell cycle as a whole is partly related to the presence of chromosome aberrations in the cell. This is demonstrated by: (i) the presence of a larger number of chromosome aberrations in M1 cells corresponding to sampling times longer after that of irradiation; (ii) the presence of a larger number of chromosome aberrations in cells with a longer G2. The most significant chromosome aberrations in this respect are isochromatid fragments. Lastly, we observed that irradiation during G1 activates another checkpoint governing the way mitosis proceeds. This takes the form of an extension of metaphase; in this case also, in some cells, activation of a possible checkpoint during preanaphase seems to be related to the presence of aberrations.