The influence of calyculin A on lymphocytes in vitro

Human lymphocytes were cultured in vitro and treated with calyculin A. The aim of this work was to estimate the influence of calyculin A on chromosome morphology and banding patterns. It was also interesting whether calyculin A treatment is useful in cytogenetic analysis of human karyotype. We proved that calyculin A induces chromosome condensation in lymphocytes and raises the mitotic index significantly. Moreover, calyculin A does not influence the banding patterns. Therefore it is concluded that calyculin A can be clinically useful for human karyotyping.     

Modulation of Serotonin Transporter Activity by a Protein Kinase C Activator and an Inhibitor of Type 1 and 2A Serine/Threonine Phosphatases

Abstract: We studied the effects of 12- O -tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC) activator, and calyculin A (CLA), an inhibitor of type 1 and 2A serine/threonine phosphatases, on serotonin uptake by a human placenta choriocarcinoma cell line (BeWo) and COS-7 cells expressing recombinant serotonin transporter (SET). In BeWo cells, treatment with TPA decreased imipramine-sensitive serotonin uptake with a reduction in V _max without affecting K _m. CLA also decreased imipramine-sensitive serotonin uptake in a manner similar to that of TPA. TPA and CLA also decreased the uptake activity of recombinant SET expressed in COS-7 cells as seen in BeWo cells. These effects of TPA and CLA were reversed by staurosporine, a protein kinase inhibitor. To elucidate whether the inhibitory effects of TPA and CLA were due to direct phosphorylation of SET by PKC, site-directed mutagenesis of five putative PKC phosphorylation sites in SET was performed. Serotonin uptake was also down-regulated by TPA and CLA in all nine mutants, suggesting that these inhibitory modulation of SET activity did not act via direct phosphorylation of SET by PKC.     

Involvement of protein phsophatase-1 in cytoskeletal organization of cultured endothelial cells

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Early replication banding reveals a strongly conserved functional pattern in mammalian chromosomes

One of the best documented autosomal linkage associations in man is on chromosome 1p and in the mouse on chromosome 4. On mitotic chromosomes this genetic homology is shown more clearly by early replication banding (RBG; induced by incorporation of 5bromodeoxyuridine (BrdU) in the second half of the S phase) than by structural banding (induced on prefixed chromosomes by denaturation, RFA, or trypsin, GTG). To analyse this phenomenon in more detail, 11 chromosomal regions in man and the domestic cat with known genetic homology were compared. In four chromosome pairs RBG and GTG banding show the same degree of homology. In seven chromosome pairs the homology is more pronounced by RBG than by GTG banding. RFA banding does not reveal the same extent of homology as does RBG banding. These results clearly show a difference between the structural banding pattern, RFA and GTG, and the replication banding pattern, RBG. The following conclusions can be drawn: in chromosomal regions with homologous functions the DNA replicates in the same temporal order. Early replication banding (RBG) reveals a functional pattern in these regions which has been more strongly preserved during evolution than the underlying chromosomal DNA. Differences in chromosomal banding are most prominent in the GTG banding pattern, whereas similarities are most apparent in the RBG banding pattern.     

Triploidy in a fetus following amniocentesis referred for maternal serum screening test at second trimester

Amniocentesis was carried out at 17 weeks gestation in a 27-year-old woman, following an abnormal maternal serum screening (MSS) test. MSS test was carried out primarily to estimate the risk of trisomy for chromosome 21. The maternal serum markers used were alpha-fetoprotein (AFP), human chorionic gonadotrophin (hCG), and unconjugated estriol (uE3), together with maternal age. The fetus was identified as screen-positive for Edward's syndrome (trisomy 18), with low uE3, normal AFP and hCG levels. The calculated risk for trisomy 18 was more than 1:50. To identify any possible chromosomal abnormality, cytogenetic investigation was carried out on the amniotic fluid sample. The fetus's karyotype showed triploidy with 69, XXX chromosome complement in all the metaphase spreads obtained from three different cultures, using GTG banding technique. Upon termination of the fetus, gross abnormalities indicative of triploidy were present in the fetus.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

同步化技术在草鱼染色体显带中的应用及草鱼核型的初步分析

对草鱼细胞进行同步化处理,并在DNA复制的早期和晚期分别掺入BrdU,制备的染色体标本置于CaCl_2溶液中温浴同时用紫外线照射,然后用Giemsa染色即可分别显示出G带和R带。标本中的部分晚前期和早中期分裂相具有高分辨染色体显带特征。用这一技术对草鱼每条染色体进行识别和分析,提出了初步的草鱼核型,发现了四对草鱼染色体具有随体。     

Endogenous sex hormones affect the mutagen-induced chromosome damage by altering a caffeine-sensitive checkpoint

In the present study we analysed the effect of endogenous sex hormones on the SCE frequencies induced in vitro by mitomycin C (MMC), a bifunctional alkylating agent producing high chromosome damage and mitotic arrest. The analysis has been performed on lymphocytes obtained at three different phases of menstrual cycle, from women with regular cycle and hormones dosage. At all phases we further analysed the effect of a post-treatment with caffeine, an agent that it is known to overrride the DNA damage checkpoints.

After MMC, the cultures obtained at ovulation and luteal phases have SCE frequencies statistically higher than the cultures obtained at the progestogenic phase, showing increases of 15 and 25%, respectively. After caffeine, the MMC treated cultures which were set up at the progestogenic phase show a high potentiation of SCE frequencies (28%) whereas the treated cultures set up at ovulatory and luteal phases show little or no potentiation.

These findings demonstrate that the endogenous hormones greatly modulate the SCE frequencies induced by the mutagen; they also indicate that hormones action competes with the caffeine effect. Caffeine acts by abrogating the mitotic arrest produced by DNA damage and induced cells with a higher chromosome damage into a premature mitosis. Our findings suggest that endogenous hormones could overcome the checkpoint controls activated in cells after mutagenic exposure. This action may be an epigenetic mechanism relevant in hormone carcinogenesis.     

Calyculin A causes the activation of histone H1 kinase and condensation of chromosomes in unfertilized sea urchin eggs independently of the maturation-promoting factor

Calyculin A is known to inhibit the type-1 and type-2A phosphatases. We previously reported that calyculin A induces contractile ring formation in unfertilized sea urchin eggs, an increase in histone H(1) kinase activity, and chromosome condensation in the calyculin A-treated unfertilized eggs, and the changes induced by calyculin A are not affected by emetine, an inhibitor of protein synthesis. These observations suggest that the mechanism by which histone H(1) kinases are activated by calyculin A is different from that of maturation-promoting factor (MPF), which is activated by a molecular modification of existed cdc2 and newly synthesized cyclin B. We report here that no cyclin B was detected by immunoblotting of unfertilized calyculin A-treated eggs. In addition, no DNA synthesis was induced by calyculin A. As well, butyrolactone I (an inhibitor of cdc2 and cdk2 kinase) had no effect on the increase in histone H(1) kinase activity nor the chromosome condensation, both of which were induced by calyculin A. Thus, we conclude that calyculin A induces histone H(1) phosphorylation in an MPF-independent manner through inhibition of type-1 phosphatase, and that the chromosome condenses as a result of histone H(1) phosphorylation.     

Platelet talin is phosphorylated by calyculin A

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.     

Partial trisomy 14q

Summary A dysmorphic female born with partial trisomy of the proximal segment of the long arm of chromosome 14 had 47 chromosomes. The extra one was acrocentric, smaller than the D group, and bigger than the G-chromosome group. By GTG banding it was identified as a deleted chromosome 14, the karyotype being 47,XX,+del 14(q24). Chromosome analysis of the parents was normal.     

FISH analysis comparing genome organization in the domestic horse (Equus caballus) to that of the Mongolian wild horse (E. przewalskii)

Przewalski's wild horse (E. przewalskii, EPR) has a diploid chromosome number of 2n = 66 while the domestic horse (E. caballus, ECA) has a diploid chromosome number of 2n = 64. Discussions about their phylogenetic relationship and taxonomic classification have hinged on comparisons of their skeletal morphology, protein and mitochondrial DNA similarities, their ability to produce fertile hybrid offspring, and on comparison of their chromosome morphology and banding patterns. Previous studies of GTG-banded karyotypes suggested that the chromosomes of both equids were homologous and the difference in chromosome number was due to a Robertsonian event involving two pairs of acrocentric chromosomes in EPR and one pair of metacentric chromosomes in ECA (ECA5). To determine which EPR chromosomes were homologous to ECA5 and to confirm the predicted chromosome homologies based on GTG banding, we constructed a comparative gene map between ECA and EPR by FISH mapping 46 domestic horse-derived BAC clones containing genes previously mapped to ECA chromosomes. The results indicated that all ECA and EPR chromosomes were homologous as predicted by GTG banding, but provide new information in that the EPR acrocentric chromosomes EPR23 and EPR24 were shown to be homologues of the ECA metacentric chromosome ECA5.     

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

Summary A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.     

玉米染色体G—带带型的研究

本文对3个玉米自交系,及其中两个自交系的杂交F_1有丝分裂早中期染色体的G-带带型进行了比较研究。所有的供试材料G-显带的染色体上都具有两种类型的带纹,我们称A型带和B型带。A型带为沿染色体长轴分布,较细的,密切邻近的多重带纹。不同自交系的A型带带型基本相同,杂交F_1的A型带无明显的异型性。非同源染色体间带型各不相同,某些染色体具有易于识别,特征性较强的A型带标记。B型带一般为深染色的大带,位于染色体的近端区。同一自交系每两个同源染色体的B型带可以配对,不同自交系B型带带型互有不同。杂交F_1某些染色体上的B型带带型异型性明显。具异型性的染色体对中一成员的带型与一个亲本相似,另一成员与另一亲本相似。比较对同一细胞先后作G-和C-显带处理的结果表明,B型带和C-带是相同的。     

Comparative cytotoxic effects of two protein phosphatase inhibitors, okadaic acid and calyculin A, on thyroid cells

The cytotoxicity of two inhibitors of protein phosphatases PP1 and PP2A has been investigated on primary cultures of dog thyrocytes. Both compounds, okadaic acid and calyculin A elicited dose- and time- related effects, i.e. apoptosis and necrosis. In addition a pronounced detachment of the cells from the monolayer was also observed. Based on the different patterns of morphological alterations and on the biochemical data, it was concluded that each compound induced different types of cell death; this provides additional evidence that a specific cell type can initiate distinct programs of death depending on the triggering stimulus. To explain the effects recorded when both compounds were added concomitantly, a functional interaction between PP1 and PP2A has been proposed. Finally, all the effects appeared modulated, to different extent, by cycloheximide and by actinomycin D. This supports the view that de novo RNA synthesis is required for the induction of death by these phosphatase inhibitors in these cells.     

Effects of antagonists of protein phosphatases on superoxide release by neutrophils.

Neutrophils stimulated with 4 beta-phorbol 12-myristate 13-acetate (PMA) release large quantities of superoxide (O2-) and exhibit phosphorylation of two proteins with molecular masses of 47(p47) and 49 kDa (p49). Addition of inhibitors of protein kinases (e.g. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) to these cells after stimulation with PMA results in the loss of 32P from these proteins and a rapid cessation of O2- release (e.g. Heyworth, P. G., and Badwey, J. A. (1990) Biochim. Biophys. Acta 1052, 299-305). In this paper we report that antagonists of type 1 and 2A protein phosphatases (okadaic acid, calyculin A) prevented both the loss of 32P from p47 and the termination of O2- release in stimulated neutrophils treated with H-7. Calyculin A also caused a remarkable hyperphosphorylation of a number of proteins in neutrophils and increased O2- release from these cells in response to a suboptimal amount of PMA. Enzymes present in both the soluble and particulate fractions of neutrophils catalyzed the near complete dephosphorylation of 32P-labeled p47 and p49 bound to Immobilon-P membranes. Dephosphorylation of these blotted phosphoproteins occurred at physiological rates and was inhibited by okadaic acid and calyculin A. These data strongly suggest that p47 undergoes a continual cycle of phosphorylation and dephosphorylation throughout the period of O2- release when PMA is the stimulus. Moreover, we show that antagonists of type 1 and 2A protein phosphatases block dephosphorylation of p47 both in vivo and in vitro, indicating that these enzymes may modulate O2- release under certain circumstances.     

Detection of BrdUrd incorporation in mammalian chromosomes by a BrdUrd antibody

Summary A bromodeoxyuridine antibody staining technique (BAT) was applied for the analysis of human chromosomes of different chromosomal band resolution. For this purpose lymphocyte cultures were synchronized and labeled with bromodeoxyuridine during the second half of the S-phase. Generally BAT was found comparable to GTG banding though some prominent GTG bands and the constitutive heterochromatin exhibit less intense staining with this technique.     

The DNA-based structure of human chromosome 5 in interphase

In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.     

Activation of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange by protein phosphatase inhibitors in red blood cells of the frog<Emphasis Type="Italic"> Rana ridibunda</Emphasis>

The present study was designed to evaluate the role of protein phosphatases in regulation of sodium transport in the marsh frog erythrocytes using 22Na as a tracer. For this purpose the cells were treated with several known inhibitors of protein phosphatases. In standard isotonic medium, exposure of the cells to 10 mmol l(-1) NaF, 20 nmol l(-1) calyculin A or 0.1 mmol l(-1) cantharidin resulted in a significant (1.7-fold) increase in unidirectional ouabain-insensitive Na+ influx. The Na+ influx in frog red cells was progressively activated as the medium osmolality was increased by addition of 100, 200 or 300 mmol l(-1) sucrose to standard isotonic medium. The stimulatory effect of protein phosphatase blockers on Na+ influx was much higher in hypertonic medium containing 100 or 200 mmol l(-1) sucrose than that in isotonic medium. Stimulation of Na+ transport enhanced with increasing concentrations of calyculin A, and half-maximal activation (EC50) was obtained at 16 nmol l(-1). However, Na+ influx induced by strong hypertonic treatment (+300 mmol l(-1) sucrose) was not altered further in the presence of protein phosphatase inhibitors. The changes in Na+ influx evoked by protein phosphatase inhibitors and hypertonic treatment were associated with a rise in the intracellular Na+, but not K+, content. Enhancement in Na+ influx after addition of protein phosphatase blockers to cell suspension in isotonic or hypertonic media was almost completely inhibited by Na+/H+ exchange inhibitors, amiloride and ethyl-isopropyl-amiloride. The basal Na+ influx in frog erythrocytes in isotonic medium was relatively low (1.7 mmol/l cells/h) and not affected by 1 mmol l(-1) amiloride. Thus, the data obtained clearly indicate that Na+/H+ exchanger in the marsh frog red blood cells is under tight regulatory control, in all likelihood via protein phosphatases of types PP-1 and PP-2A.