Premature chromosome condensation. II. The nature of chromosome gaps produced by alkylating agents and ultraviolet light

Chinese hamster ovary (CHO) cells were treated with ultraviolet radiation or the alkylating agents, nitrogen mustard or trenimon, and chromosome damage to G2 phase cells were scored by the premature chromosome condensation (PCC) method or the metotic chromosome method. Treatment with these agents produced gaps but not chromatid breaks or exchanges. After UV treatment, the gap frequency observed in G2-PCC was higher than in the mitotic chromosomes, while the reverse trend was observed after treatment with nitrogen mustard or trenimon. These results suggest that two types of chromosome gaps exist, both of which are observable in mitotic chromosomes while only one type is observable in PCC due to differences in the stages of condensation between PCC and mitotic chromosomes.     

Role of polyamines during chromosome condensation of mammalian cells

The objective of the present study was to investigate the role of polyamines in the process of chromosome condensation. The phenomenon of premature chromosome condensation (PCC) involving fusion between mitotic and interphase cells was used as the assay system. The factors present in the mitotic cells would bring about the breakdown of the nuclear membrane and condensation of the interphase chromatin into chromosomes, similar to that which occurs during the initiation of mitosis. Alpha-difluoromethyl ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase was used to deplete both mitotic and interphase cells of polyamines. The results indicate that the polyamine depleted mitotic cells have a diminished ability to induce PCC. This inhibition could easily be reversed by exogenous addition of polyamines at the time of fusion. Furthermore, exogenously added polyamines hastened the entry of exponentially growing cells into mitosis. These observations suggest an essential role for polyamines during the process of chromosome condensation of mammalian cells.     

Analysis of X-ray induced aberrations in mammalian chromosomes by electrofusion induced premature chromosome condensation

Summary Premature chromosome condensation (PCC) was induced by electrofusion of metaphase cells of an Ehrlich ascites tumor cell line with interphase cells of a Muntjac cell line or of a Chinese Hamster subline. Electrofusion was performed by cell alignment in a weakly inhomogeneous a.c. field of 200 V/cm amplitude (peak-to-peak value) and of 1.7 MHz frequency, followed by the application of a series of breakdown (fusion) pulses of 5 kV/cm strength and 15 µs duration. Most of the PCC's were of the G2 type despite the large proportion of G1 and S cells in the suspension. The number of chromatid aberrations observed in electrofused cells which had not been subjected to irradiation was not significantly above the spontaneous level. This indicates that electrofusion, at least as used here, did not lead to lesions expressed as structural aberrations. When interphase cells were irradiated by X-ray doses below 3 Gy before electrofusion PCC analysis showed chromosome damage consisting mainly of breaks and gaps. The frequency of aberrations recorded by PCC was 6 to 40 fold larger than that seen in conventional metaphase analysis. This large increase probably arose because of an effective suppression of the G2 repair of chromosomal lesions by the fast condensation process which took place within about 30 min. This assumption was supported by PCC experiments in which the time between X-irradiation and fusion with subsequent chromosome condensation was varied. The results demonstrated that G2 repair of chromosomal lesions was not detectable until 20 min after fusion with a half-time of the repair kinetics of about 1.5 h. The selectivity of premature chromosome condensation in G2 cells is discussed in terms of the differences between electrofusion and chemically or virally induced fusion. It is assumed that the concentration and the transfer rate of the chromosome condensation factor from the metaphase to the interphase cell are the limiting factors in achieving PCC. This is because the localised permeabilisation of the membrane and the dominance of two-cell fusions are characteristic of electrofusion.     

Chromosome condensation outside of mitosis: mechanisms and new tools

A basic principle of cell physiology is that chromosomes condense during mitosis. However, condensation can be uncoupled from mitotic events under certain circumstances. This phenomenon is known as "premature chromosome condensation (PCC)." PCC provides insights in the mechanisms of chromosome condensation, thus helping clarifying the key molecular events leading to the mitosis. Besides, PCC has proved to be an useful tool for analyzing chromosomes in interphase. For example, using PCC we can visualize genetic damage shortly after the exposure to clastogenic agents. More than 30 years ago, the first report of PCC in interphase cells fused to mitotic cells using Sendai virus was described (virus-mediated PCC). The method paved the way to a great number of fundamental discoveries in cytogenetics, radiation biology, and related fields, but it has been hampered by technical difficulties. The novel drug-induced PCC method was introduced about 10 years ago. While fusion-induced PCC exploits the action of external maturation/mitosis promoting factor (MPF), migrating from the inducer mitotic cell to the interphase recipient, drug-induced PCC exploits protein phosphatase inhibitors, which can activate endogenous intracellular MPF. This method is much simpler than fusion-induced PCC, and has already proven useful in different fields.     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.     

Premature chromosome condensation and cell cycle analysis.

The application of the phenomenon of premature chromosome condensation for cell cycle analysis in HeLa and CHO cells has been examined. Random populations of HeLa and CHO cells pulse labelled with H3-TdR were separately fused with mitotic HeLa cells using U.V. inactivated Sendai virus. The resulting prematurely condensed chromosomes (PCC) were scored and classified into G1, S and G2-PCC on the basis of both morphological and autoradiographic data, The results of this study indicated that the G1, S and G2 phase cells are equally susceptible to virus-induced fusion with mitotic cells and subsequent induction into PCC. Hence the PCC method for cell cycle analysis is both practical and accurate. This study also revealed that the process of chromosome decondensation initiated during the telophase of mitosis continues throughout the G1 period reaching an ultimate state of decondensation by the end of G1, at which point the fusion of such cells with those in mitosis yield PCC with the most diffused morphology instead of the discrete single stranded structures characteristic of early G1-PCC. Thus, the decondensation of chromatin during G1 appears to be a prerequisite for the subsequent initiation of DNA synthesis.     

Relationship between histone phosphorylation and premature chromosome condensation

The relationship between histone phosphorylation and chromosome condensation was investigated by determining changes in phosphorylation levels of histones H1 and H3 following fusion between mitotic and interphase cells and subsequent premature chromosome condensation. We detected significant increases in the levels of phosphorylation of H1 and H3 from interphase chromatin in which a majority of nuclei had undergone premature chromosome condensation. In addition, we noted significant decreases in the phosphate content of the highly phosphorylated mitotic H1 and H3, presumably resulting from phosphatase activity contributed by the interphase component of mitotic/interphase fused cells. These observations further strengthen the correlation between histone phosphorylation and the changes in chromosome condensation associated with the initiation of mitosis. This study also suggests that maintenance of the mitotic chromosomes in a highly condensed state does not require the continued presence of histones in a highly phosphorylated form.     

Mammalian cell fusion. IV. Regulation of chromosome formation from interphase nuclei by various chemical compounds

Hybrid HeLa cells formed by the fusion of mitotic with interphase cells have been used as a test system to study the effects of various positively and negatively charged compounds on the induction of premature chromosome condensation (PCC) of the interphase nuclei. Among the various positively charged compounds tested, spermine, putrescine and Mg⁺⁺ were specific in promoting the PCC induction while spermidine was unique in inhibiting this event. All the negatively charged compounds including estradiol-17β were uniformly inhibitory. The inhibitory effect of estradiol was reversed by putrescine. The inhibition of PCC induction by estradiol seems to be due to its binding to interphase chromatin rather than to the PCC inducers. The differences between the polyamines in their effects on the PCC inducing system has been explained on the basis of their abilities to bind stereospecifically with chromatin.     

Changes in the organization of chromosomes during the cell cycle: response to ultraviolet light.

Fusion between mitotic and interphase cells results in the premature condensation of the interphase chromosomes into a morphology related to the position in the cell cycle at the time of fusion. These prematurely condensed chromosomes (PCC) have been used in conjunction with u.v. irradiation to examine the interphase chromosome condensation cycle of HeLa cells. The following observations have been made: (I) There is a progressive decondensation of the chromosomes during G1 which is accentuated by u.v. irradiation: (2) The chromosomes become more resistant to u.v.-induced decondensation during G2 and mitosis. (3) There is a close correlation between the degree of chromosome decondensation and the amount of unscheduled DNA synthesis induced by u.v. irradiation during G1 and mitosis: (4) Hydroxyurea enhances the ability of u.v. irradiation to promote the decondensation of chromosomes during G1, G2 and mitosis. Hydroxyurea also potentiates the lethal action of u.v. irradiation during mitosis and G1. These data are discussed in relation to the suggestion that chromosomes undergo a progressive decondensation during G1 and condensation during G2.     

Perturbation of mammalian cell division. II. Studies on the isolation and characterization of human mini segregant cells.

A method is described for the isolation, according to size, of mini segregants produced by the abnormal cleavage of reversibly arrested mitotic HeLa cells. Many of these mini segregants contain small amounts of DNA, as judged by Feulgen staining and chromosome analysis. After fusion with mitotic HeLa cells, the interphase chromosomes of the mini segregants are seen as either monovalent or bivalent prematurely condensed chromosomes (PCC), some of which are damaged. A proportion of isolated mini segregants synthesize DNA, RNA and protein. Fusion of mini segregants with interphase HeLa cells gives rise to cells with 'hybrid' karyotypes.     

The centriolar cycle in polykaryons containing prematurely condensed chromosomes or telophase-like nuclei]

In fused interphase-mitotic cells, either interphase nuclei are induced to premature chromosome condensation (PCC) or mitotic chromosomes are induced to telophase-like nuclei (TLN) formation. This study concerns structural and functional changes in centrioles of fused cells in which PCC or TLN are induced. Embryonic pig kidney cells were fused using a modified PEG-DMSO-serum method. Cell cycle period of the nuclei was determined before cell fusion using double-labeling autoradiography. Polykaryons containing desirable type of PCC or interphase nuclear combination in TLN were selected on the basis of isotope labeling after being embedded in epon. Selected cells were cut into serial sections and studied under electron microscope. The data obtained showed that centrioles at every interphase period undergo mitotic activation when their nuclei are induced to PCC. They acquire fibrillar halo and form half-spindles. Daughter centrioles at G1, S and G2 periods are also capable of mitotic activation when separated from their mother centriole. Inert centrioles were found in some cells with G1-PCC. When mitotic nuclei are induced to TLN formation, their centrioles also become inactivated. They lose fibrillar halo and mitotic spindles break down. Some mitotic centrioles develop features characteristic of interphase period such as satellites and vacuoles. Induced nuclear and centriolar changes are simultaneous and may be controlled by the same factor. Mitotic factor of mitotic cell partner which induces PCC may also induce interphase centrioles to mitotic activation. Degradation of the mitotic factor leading to TLN formation may also cause the loss of the mitotic activity of centrioles and disorganization of mitotic spindles.     

Radiation-induced cytogenetic damage in relation to changes in interphase chromosome conformation

The premature chromosome condensation (PCC) technique was used to study several factors that determine the yield of chromosome fragments as observed in interphase cells after irradiation. In addition to absorbed dose and the extent of chromosome condensation at the time of irradiation, changes in chromosome conformation as cells progressed through the cell cycle after irradiation affected dramatically the yield of chromosome fragments observed. As a test of the effect of chromosome decondensation, irradiated metaphase Chinese hamster ovary (CHO) cells were allowed to divide, and the prematurely condensed chromosomes in the daughter cells were analyzed in their G1 phase. The yield of chromosome fragments increased as the daughter cells progressed toward S phase and chromosome decondensation occurred. When early G1 CHO cells were irradiated and analyzed at later times in G1 phase, an increase in chromosome fragmentation again followed the gradual increase in chromosome decondensation. As a test of the effect of chromosome condensation, G0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. It can be concluded that changes in chromosome conformation interfered with rejoining processes. In contrast, resting chromosomes (as in G0 lymphocytes irradiated before fusion) showed efficient rejoining. These results support the hypothesis that cytogenetic lesions become observable chromosome breaks when chromosome condensation or decondensation occurs during the cell cycle.     

Induction by chemical clastogens of aberrations in prematurely condensed interphase chromatin of Chinese hamster ovary cells

The clastogenic activities of diepoxybutane and bleomycin were comparatively studied on prematurely condensed interphase chromatin and metaphase chromosomes of Chinese hamster ovary cells. The yield of chromosomal aberrations was distinctly higher in G2-premature chromosome condensation as compared to metaphase. Most notably, the clastogenic activity of bleomycin was visible in premature chromosome condensation after application of much lower final concentrations than necessary for induction of chromosome aberrations in metaphase. In addition, the different mechanisms of action of both clastogens were reflected by the aberration yield in GI and G2 immediately after exposure. While bleomycin induced aberrations throughout all stages of interphase, diepoxybutane did not induce aberrations in GI or G2. Though certainly not a routine system for genotoxicity testing, premature chromosome condensation analyses provide a powerful opportunity to demonstrate relationships between DNA damage and repair, and the production of chromosomal changes at the site of their formation.Abbreviations BM bleomycin - BrdUrd bromodeoxyuridine - CHO Chinese hamster ovary - DEB diepoxybutane - DMSO dimethylsulfoxide - FCS fetal calf serum - PCC premature chromosome condensation, prematurely condensed chromosomes - PEG polyethylene glycol     

High-resolution measurement of breaks in prematurely condensed chromosomes by differential staining

A relatively simple method has been developed to improve the resolution for measuring breaks produced in interphase chromosomes by X rays or other agents following the induction of premature chromosome condensation (PCC). Mitotic HeLa cells, which induce PCC when fused with interphase cells, were obtained from cultures grown for several generations in 5-bromodeoxyuridine (BrdU). These were fused to cells from low-passage confluent cultures of normal human fibroblasts and subsequently stained by a modified fluorescence-plus-Giemsa (FPG) technique. Following this protocol the prematurely condensed chromosomes stain intensely, whereas the mitotic chromosomes of the inducer cell(s), which are intermingled with them, stain very lightly. With this technique the interphase chromosomes and their fragments can be identified unequivocally, making scoring much easier and more accurate. The frequency of breaks produced in G1 phase AG1522 human fibroblasts immediately following X-ray doses of 58 and 117 rad was 3.68 and 7.38 per cell, respectively. Use of this technique should allow the detection of damage from ionizing radiation at doses lower than 10 rad.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique

The complementation effect of wild-type CHO-K1 and xrs mutants after fusion, as judged by the frequencies of X-ray-induced G1 and G2 premature chromosome condensation (PCC), was studied. For induction of PCC, X-irradiated interphase cells (G1 and G2) were fused immediately with untreated mitotic cells of the same cell line or with mitotic cells of another line. The frequencies of breaks in G1-PCC, or breaks and chromatid exchanges in G2-PCC were determined and the latter parameter was compared with the frequency of chromosomal aberrations in mitotic cells following G2 irradiation. CHO-K1 cells were capable of complementing the X-ray sensitivity of both xrs 5 and xrs 6 cells. However, full restoration of the repair defect in xrs cells could never be accomplished. The mutants failed to complement each other. In CHO-K1 cells, the incidence of chromosomal aberrations was significantly higher in G2-PCC (2.5-fold) than that observed in mitotic cells at 2.5 h after irradiation. The ratio of the induced frequency of aberrations in G2-PCC to that in mitotic cells was correlated with the degree of repair of DNA double-strand breaks (dsb) and reached almost 1 in xrs 5 cells indicating no repair. In addition the data indicated that, during the period of recovery of CHO-K1 cells, X-ray-induced breaks decreased but exchanges remained at the same level. In contrast, due to a deficiency in rejoining of dsb in xrs mutants, breaks remained open for a long period of time, allowing the formation of additional chromatid exchanges during recovery time.     

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.     

Antibodies specific for mitotic human chromosomes

The induction of premature chromosome condensation in an interphase cell immediately following fusion with a mitotic cell suggests the presence of factors in the mitotic cell that are responsible for the transformation of an interphase nucleus into prematurely condensed chromosomes (PCC). Several lines of evidence suggest that these factors are proteins present in the cytoplasm of mitotic cells. The objective of this study was to raise antibodies to the factors responsible for PCC. Cytosol from synchronized mitotic HeLa cells was injected into rabbits in order to obtain antiserum. The IgG fraction from this antiserum reacted with 98% of mitotic HeLa cells when tested by indirect immunofluorescence. Most of the fluorescence was localized on the chromosomes. About 5% of the interphase nuclei also reacted with the antiserum, but 50% of these cells were in early G1. Antigenic reactivity was induced in the condensing interphase chromatin in 31% of the interphase nuclei found in mitotic-interphase fused cells. Rodent cells did not react with the antibody by indirect immunofluorescence. Mitotic HeLa cells were able to induce antigenic reactivity in 23 % of interphase Chinese hamster ovary (CHO) cell nuclei in fused binucleate cells, whereas the converse was not true of mitotic CHO cells. Enzyme digestion and incubation with denaturing agents suggested that antigenic reactivity depended on a DNA-non-histone protein complex.     

Nuclear lamins during prophasing and telophasing in heterophasic HeLa and Chinese hamster homokaryons

We have perturbed the dynamics of the nuclear lamins by means of cell fusion between mitotic and interphase cells and have studied redistribution of lamins in fused cells as a function of extracellular pH levels. We show here that in heterophasic M-1 HeLa homokaryons disassembly of interphase lamins predominates at low pH levels between 7.0 to 7.3, whereas deposition of cytoplasmic lamins around condensed metaphase chromosomes was observed at pH 8.0. In HeLa homokaryons lamina disassembly and lamina deposition around chromosomes are mutually exclusive. Using heterophasic M-1 homokaryons of the Chinese hamster cell line DON we observed that disassembly of interphase lamins and deposition of lamins around condensed chromosomes coexisted in the same homokaryon kept at pH 7.0. Disassembly of lamins developed synchronously with premature chromosome condensation (PCC) whereas lamina deposition around the condensed M-chromosomes was followed by telophasing. In fusions kept at pH 8.0 cytoplasmic lamins were exclusively deposited around mitotic chromosomes. The results are interpreted as showing that pH regulates the lamina dynamics in homokaryons of mitotic and interphase cells.     

Reversible and irreversible inhibitors of clustering of alpha 2M in clathrin-coated pits on the surface of fibroblasts

An efficient polyethylene glycol-mediated fusion procedure has been developed and standardised for induction of premature chromosome condensation at high frequency in HeLa cells. High fusion frequencies are achieved on a dense cell monolayer induced by centrifugation of mitotic and interphase cells onto lectin-coated plastic culture dishes. Using this fusion procedure, induction frequencies of 60–90 % can routinely be obtained. This method should be useful in inducing sufficient quantities prematurely condensed chromosomes (PCC) for purification and subsequent biochemical and ultrastructural analysis. The procedure can be readily adapted for other studies that require high fusion frequencies between non-attaching cell types.