Abnormal Chromatin Condensation Research Articles

Effects of protein kinase and phosphatidylinositol-3 kinase inhibitors on growth and ultrastructure of Trypanosoma cruzi

An increasing number of protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. Some PK inhibitors display antiproliferative effects on protozoa. We tested three PK inhibitors on the growth and ultrastructure of epimastigotes of Trypanosoma cruzi and the effect of these drugs on intracellular amastigotes. They were staurosporine (serine/threonine kinase inhibitor), genistein (tyrosine kinase inhibitor), and wortmannin (phosphatidylinositol 3' (PI3) kinase inhibitor). All drugs inhibited epimastigote growth at the concentrations tested. Wortmannin inhibited parasite growth at the lowest concentrations. However, staurosporine was the most effective after 24 h treatment and genistein caused the stronger inhibition during the whole treatment (60-70% inhibition). The IC50 were: staurosporine: 6.43+/-1.28 microM; genistein: 6.54+/-1.86 microM; and wortmannin: 0.056+/-0.014 microM. These PK inhibitors had strong ultrastructural effects on the epimastigotes: abnormal chromatin condensation of the nucleus; loose flagellar membrane with the formation of blebs; incomplete cell division; autophagosomes and myelin-like figures. These drugs did not interfere with the division of intracellular amastigotes or with its differentiation to trypomastigotes. However, as trypanosomes have kinomes that contain a large set of protein kinases and phosphatases, PKs should not be disregarded as an important target for chemotherapy of Chagas disease.

Effect of a β-lapachone-derived naphthoimidazole on Trypanosoma cruzi: identification of target organelles

Investigation of the mode of action of the naphthoimidazole N1, obtained from the reaction of beta-lapachone with benzaldehyde, which among 45 semi-synthetic derivatives of naphthoquinones isolated from Tabebuia sp. was one of the most active compounds against Trypanosoma cruzi trypomastigotes. Quantification of the effect of N1 against the proliferative forms of T. cruzi, and investigation of potential targets in the parasite using electron microscopy and flow cytometry techniques. N1 presented the following order of activity: amastigotes > trypomastigotes > epimastigotes. The effect on intracellular forms was approximately 25 times higher than on macrophages and heart muscle cells. N1-treated parasites presented an abnormal chromatin condensation and mitochondrial damage. In epimastigotes, alterations of reservosomes were observed, and in trypomastigotes, a decrease in the electron density of acidocalcisomes was observed. In epimastigotes, the naphthoimidazole inhibited the activity of succinate cytochrome c reductase. Labelling with rhodamine 123 or Acridine Orange was decreased in both forms treated with N1. The results suggest that epimastigotes, reservosomes, mitochondrion, and nucleus contain N1 targets. In trypomastigotes, in which reservosomes are absent, the organelles affected by the compound were also the mitochondrion and nucleus, as well as acidocalcisomes, in which the decrease in electron density could be due to the use of polyphosphate as an alternative energy supply.

Identification and Characterization of SSTK, a Serine/Threonine Protein Kinase Essential for Male Fertility

Here we describe and characterize a small serine/threonine kinase (SSTK) which consists solely of the N- and C-lobes of a protein kinase catalytic domain. SSTK protein is highly conserved among mammals, and no close homologues were found in the genomes of nonmammalian organisms. SSTK specifically interacts with HSP90-1beta, HSC70, and HSP70 proteins, and this association appears to be required for SSTK kinase activity. The SSTK transcript was most abundant in human and mouse testes but was also detected in all human tissues tested. In the mouse testis, SSTK protein was localized to the heads of elongating spermatids. Targeted deletion of the SSTK gene in mice resulted in male sterility due to profound impairment in motility and morphology of spermatozoa. A defect in DNA condensation in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins. SSTK phosphorylated histones H1, H2A, H2AX, and H3 but not H2B or H4 or transition protein 1 in vitro. These results demonstrate that SSTK is required for proper postmeiotic chromatin remodeling and male fertility. Abnormal sperm chromatin condensation is common in sterile men, and our results may provide insight into the molecular mechanisms underlying certain human infertility disorders.

SDX-105 (Treanda™), Active in Non-Hodgkin's Lymphoma Cells, Induces the Mitotic Catastrophe Death Pathway.

SDX-105 (Treanda™) is an alkylating agent with a distinct mechanism of action that has been shown to be active in clinical trials in NHL and CLL patients refractory to traditional DNA-damaging agents. SDX-105 induces unique changes in gene expression in NHL cells and displays a lack of cross resistance with other 2-chloroethylamine alkylating agents. Quantitative PCR analysis confirmed that the G2/M checkpoint regulators Polo-like kinase 1 (PLK-1) and Aurora A kinase (AurkA) are down-regulated in the NHL cell line SU-DHL-1 after 8 hours of exposure to clinically relevant concentrations of SDX-105. No changes in these same genes were observed when cells were exposed to equi-toxic doses of chlorambucil or an active metabolite of cyclophosphamide. Because our previous studies demonstrated that SDX-105 treatment can activate apoptotic cell death pathways, we examined the ability of SDX-105 to induce cytotoxicity in cells unable to undergo ‘classical' caspase-mediated apoptosis. Multi-drug resistant MCF-7/ADR cells and p53 deficient RKO-E6 colon adenocarcinoma cells were exposed for two or three days to either 50 μM SDX-105 alone or 50 μM SDX-105 and 20 μM pan-caspase inhibitor zVAD-fmk. Although zVAD-fmk was able to inhibit SDX-105 induced increases in Annexin-V-positive cells, microscopic analysis of nuclear morphology using the DNA stain DAPI in cells treated with either SDX-105 alone or in combination with zVAD-fmk showed increased incidence of micronucleation. Multi/micro-nucleation and abnormal chromatin condensation are both hallmarks of mitotic catastrophe and have been observed in tumor cells exposed to microtubule-binding drugs such as the vinca alkaloids and taxanes. Activation of mitotic catastrophe may amplify the cytotoxicity of SDX-105 and its activity in tumor cells where classical apoptotic pathways are inhibited. This may explain, at least in part, the potent anti-tumor activity of SDX-105 in tumor cells refractory to conventional 2-chloroethylamine DNA-damaging agents. Additional studies are ongoing to further elaborate the role of mitotic catastrophe in SDX-105's mechanism of action. The capacity to induce mitotic catastrophe may explain the anti-tumor activity of SDX-105 in chemotherapy relapsed and resistant patients.

Chromatin condensation and DNA damage of human epididymal spermatozoa in obstructive azoospermia

Obstructive azoospermia (OA) includes patients with different aetiologies of obstruction such as congenital bilateral absence of vas deferens (CBAVD), post-vasectomy/failed vaso–vasostomy, or unknown (idiopathic). The frequency of DNA-damaged spermatozoa and abnormal chromatin condensation in epididymal spermatozoa of OA patients was investigated for the different aetiology subgroups. DNA damage in caput epididymis spermatozoa was assessed with the TdT-UTP nick-end labelling (TUNEL) assay and chromatin condensation status was measured with chromomycin A3 (CMA3) stain ( n = 60 patients). All epididymal sperm samples showed high levels of TUNEL positivity (mean and SD, 43 ± 17%). CMA3 rates showed large variation within the samples (mean and SD, 53 ± 21%, range 7–97%); however, a significant difference in chromatin condensation was found between the different aetiologies of obstruction. The highest percentage of CMA3 positivity was found in the CBAVD and idiopathic groups (60 ± 17 and 65 ± 18% respectively) compared with the post-vasectomy samples (37 ± 17%) or ejaculated spermatozoa from normozoospermic males (29 ± 12%). In conclusion, despite the normal testicular histology in OA men, chromatin condensation differed between OA patients. The physiological heterogeneity found in chromatin condensation between patient subgroups is probably influenced by the aetiology of the obstruction.

Caenorhabditis elegans nucleoporins Nup93 and Nup205 determine the limit of nuclear pore complex size exclusion in vivo.

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate communication between the nucleus and the cytoplasm. To obtain insight into the structure and function of NPCs of multicellular organisms, we have initiated an extensive analysis of Caenorhabditis elegans nucleoporins. Of 20 assigned C. elegans nucleoporin genes, 17 were found to be essential for embryonic development either alone or in combination. In several cases, depletion of nucleoporins by RNAi caused severe defects in nuclear appearance. More specifically, the C. elegans homologs of vertebrate Nup93 and Nup205 were each found to be required for normal NPC distribution in the nuclear envelope in vivo. Depletion of Nup93 or Nup205 caused a failure in nuclear exclusion of nonnuclear macromolecules of approximately 70 kDa without preventing active nuclear protein import or the assembly of the nuclear envelope. The defects in NPC exclusion were accompanied by abnormal chromatin condensation and early embryonic arrest. Thus, the contribution to NPC structure of Nup93 and Nup205 is essential for establishment of normal NPC function and for cell viability.

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility

This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.

Phosphatidylserine induces apoptosis in adherent cells.

Phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane in apoptotic cell death. However, the roles of PS in apoptotic signaling are still unclear. In this study, we found that exogenous PS, but not other phospholipids, induced cell death in adherent cells, but not in suspension culture. The cell death exhibited typical features of apoptosis such as cell shrinkage, nuclear fragmentation and abnormal chromatin condensation. When PS was added to CHO-K1 cells in monolayer culture, they began to show changes in cell shape and actin cytoskeleton and protein kinase C (PKC) activity, followed by cell detachment, caspase activation, cleavage of focal adhesion kinase (FAK) and finally loss of viability. These results suggested that PS causes apoptosis through actin disorganization, cell detachment and cleavage of FAK.

Distinct Chk2 Activation Pathways Are Triggered by Genistein and DNA-damaging Agents in Human Melanoma Cells

Genistein, a natural isoflavone found in soybeans, exerts a number of biological actions suggesting that it may have a role in cancer prevention. We have previously shown that it potently inhibits OCM-1 melanoma cell proliferation by inducing a G(2) cell cycle arrest. Here we show that genistein exerts this effect by impairing the Cdc25C-dependent Tyr-15 dephosphorylation of Cdk1, as the overexpression of this phosphatase allows the cells to escape G(2) arrest and enter an abnormal chromatin condensation stage. Caffeine totally overrides the genistein-induced G(2) arrest, whereas the block caused by etoposide is not bypassed and that caused by adriamycin is only partially abolished. We also report that genistein activates the checkpoint kinase Chk2 as efficiently as the two genotoxic agents and that caffeine may counteract the activation of Chk2 by genistein but not by etoposide. In contrast, caffeine abolishes the accumulation of p53 caused by all the compounds. Wortmannin does not suppress the Chk2 activation in any situation, suggesting that the ataxia telangiectasia-mutated kinase is not involved in this regulation. Finally, unlike etoposide and adriamycin, genistein induces only a weak response in terms of DNA damage in OCM-1 cells. Taken together, these results suggest that the G(2) checkpoints activated by genistein and the two genotoxic agents involve different pathways.

Extra‐epididymal spermatozoa express nuclear abnormalities

Extra-epididymal spermatozoa account for approximately a third of all spermatozoa found in the normal human ejaculate. Whilst remaining outside of the testes at core body temperature, the functional competence of spermatozoa, including cell motility and fertilizing capacity, diminishes. By examining spermatozoa found in the seminal fluid of recently vasectomized men, this study has investigated the nuclear changes that occur in spermatozoa whilst persisting in sites distal to the epididymis. Spectral recordings of spermatozoa stained with the nucleic acid dye, toluidine blue and the sperm chromatin structure assay (SCSA) were performed. Toluidine blue staining of human sperm DNA is an effective predictor of abnormal protamine disulphide crosslinking and chromatin condensation. Using flow cytometry, the SCSA determines the sensitivity of sperm DNA to acid-induced denaturation, providing a measure of chromatin and DNA damage. Abnormal protamine disulphide crosslinking and chromatin condensation was significantly higher in spermatozoa from patients after vasectomy when compared to normozoospermic controls (p < 0.01). Additionally, spermatozoa from vasectomized donors were significantly more sensitive to acid-induced denaturation than were normozoospermic donors (p < 0.05). The results indicate that spermatozoa surviving in extra-epididymal sites are more likely to possess DNA and chromatin abnormalities than those present in the testes and epididymis. These changes may partly explain the depletion of cell viability and fertilizing capacity of extra-epididymal spermatozoa which has been reported previously.

Ectopic expression of the Aspergillus nidulans mitotic inducer, nimA kinase, in megakaryocytes : Effect on polyploidization

Aspergillus nidulans nimA gene encodes a serine/threonine protein kinase (NIMA) whose activity is essential for mitotic entry and chromatin condensation. Both the activity and the abundance of NIMA protein increase at the G2/M transition of the fungal cell cycle. In this study, we report the effects elicited by ectopic expression of nimA on polyploidization in a mouse megakaryocytic line, Y10, which is undergoing an endomitotic cell cycle. A pool of Y10 stable transfectants that have been induced to express nimA displayed a decrease in cell number and an elevated DNA content per cell. NIMA also dramatically enhanced the activity of phorbal 12-myristate 13-acetate toward polyploidization. Analysis of individual nimA transfectants revealed that the DNA content per cell rose in cells expressing high levels of nimA and that the level of cyclin B was reduced as compared to the mock-transfected cells. These effects observed in polyploidizing megakaryocytes are in contrast to those found in A. nidulans and HeLa cells, in which induced nimA expression caused abnormal chromatin condensation and cell cycle arrest. We conclude that high-level expression of nimA in cells programmed to undergo endomitosis could potentiate polyploidization. The challenge now resides in the isolation of the authentic megakaryocyte counterpart of the fungal nimA.

Flow cytometric and microscopic evaluation and effect on fertility of abnormal chromatin condensation in bovine sperm nuclei.

The techniques of Feulgen staining, acridine orange staining, and a sperm chromatin structure assay using acridine orange and flow cytometry were compared for selective examination of bovine sperm nuclei. Twenty frozen semen samples were simultaneously analysed by all three methods. The prevalence of abnormally condensed DNA and its relationship to other semen traits were determined in ejaculates from 70 bulbs presented for routine examination for breeding soundness and in frozen semen from 348 bulls evaluated over five years. A breeding trial with 118 beef heifers using semen from six bulls with different degrees of nuclear abnormalities was performed to assess the importance of the defects with respect to fertility. The results indicate that few spermatozoa with abnormal DNA condensation are found in normal semen, but the incidence increases with disturbance of spermatogenesis. However, high numbers of abnormally condensed nuclei were found in the absence of an increase in other defects. This nuclear defect might be at least partially of epididymal origin; it can lower fertility and can be compensated for by increasing the numbers of normal spermatozoa in the insemination dose. The percentage of abnormally condensed sperm nuclei as detected by Feulgen staining was significantly correlated with that detected by microscopy after acridine orange staining and by the sperm chromatin structure assay. We therefore consider the Feulgen technique to be a valuable tool for assessing the nuclear integrity of bovine spermatozoa.

Sperm quality assessed by flow cytometry and accessory sex gland function in spinal cord injured men after repeated vibration-induced ejaculation

Semen was obtained by vibration-induced ejaculation from 5 spinal injured men once a week for 5 consecutive weeks under standardised conditions. The site of the spinal lesions varied from C5 to Th10. Although in all subjects except one, the total sperm count in the first ejaculate was within normal limits, conventional criteria indicated a high degree of asthenoteratozoospermia in all cases. In subsequent ejaculates there was no major general improvement in motility, vitality or morphology. However, 2 individuals exhibited a marked increase in the proportion of motile sperm in the ejaculate over the next 3 weeks. Flow cytometry of the same sperm samples indicated a high degree of abnormal chromatin condensation and reduced binding of a fluorescent acrosomal marker in the first ejaculates. No improvement in these parameters could be detected with time. Assessment of accessory sex gland function using specific secretory markers indicated that compared to the normal population, the vesicular contribution was markedly reduced in 3 subjects and prostatic contribution in the 2 remaining subjects in the first and subsequent ejaculations. Ejaculate volumes were consistently low in all subjects during the observation period. In contrast, total epididymal secretion was comparable to normal ejaculates. Prostatic contributions to the ejaculate increased significantly over the first 4 weeks. In conclusion, regular vibration-induced ejaculation at weekly intervals could not improve sperm quality in paraplegic men to an acceptable degree for assisted fertilisation to be recommended. Although certain aspects of sperm quality, as judged by conventional criteria, were improved in some cases, flow cytometry revealed persistent chromatin and acrosomal abnormalities.

Flow Cytometric Evaluation of Sperm from Patients with Testicular Carcinoma

Semen samples from 14 patients with testicular cancer were analyzed by new flow cytometry techniques and by conventional semen analysis. Samples were obtained post-unilateral orchiectomy and prior to further treatment. With only 1 exception, light microscopic analysis of sperm count and morphology indicated a decrease of normal sperm production and incomplete or abnormal differentiation. Flow cytometry of semen aliquots showed, with the same exception as above, increased acridine orange staining of the sperm nuclear chromatin, attributed to abnormal chromatin composition and condensation. In addition, the chromatin structure of isolated sperm nuclei was abnormally sensitive to thermal stress. These changes were present up to 15 months post orchiectomy and suggest abnormal spermiogenesis in the contralateral testis of patients with testicular tumors. Of the 14 patients studied only 1 fell well within the normal range for all parameters measured; 2 other patients were in the low normal range for all parameters and 1 patient fell below normal for light microscope derived measurements but was normal by flow cytometry. The other 10 patients had abnormal measurements for all parameters.The assessment of spermiogenesis by flow cytometry studies of sperm chromatin appears to be a sensitive and valuable new parameter of fertility that cannot be determined by conventional semen analysis.

Changes Induced to Bull Spermatids and Testicular Spermatozoa by a Single Peritesticular Injection of Ethylene Dibromide

Peritesticular injection of ethylene dibromide to bulls resulted in misshaping, abnormal chromatin condensation and decrease of UV‐DNA and UV‐protein contents of elongating spermatids.

The effect of ethylene dibromide on differentiation of the acrosome, nucleus, and transient nuclear appendages in ram spermatids

Protracted parenteral treatment of rams with ethylene dibromide disturbed the differentiation of the acrosome, nucleus, and transient nuclear appendages. The prime target was the acrosome, hit first and most during its growing phase, i.e., the cap phase of spermiogenesis. The results were aplasia, hypoplasia, or hyperplasia; deformation of the acrosomic cap; vesicular inclusions of the acrosomic matrix; and lesions in the acrosome attachment to the nucleus. Some of these anomalies could be related to aberrant morphology and activity of the Golgi complex. The drug also affected nuclear metamorphosis taking place in the third phase of spermiogenesis. The effect was expressed in abnormal chromatin condensation, disturbed sap elimination, and deviation from modal shaping. It is postulated that the nuclear metamorphosis is supported by a machinery consisting of the acrosome, caudal manchette, and Sertoli cell mantle. The acrosome assumes the key position since it seems to be the prerequisite for cell polarization and the polarization essential for differentiation of the caudal manchette and Sertoli cell mantle. The absence of the acrosome accounted for a complete failure of nuclear shaping, while the lack of other appendages accounted for a partial deviation from the modal nuclear shape.