Comparison of the major polypeptides of the erythrocyte nuclear envelope

The three most abundant nonhistone polypeptides (molecular weights 75,000, 71,000 and 61,000) of the avian erythrocyte nucleus have previously been isolated in the nuclear envelope fraction. They have been separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and peptide-mapped after limited enzymatic digestion. Three enzymes–chymotrypsin, papain and Staphylococcus aureus protease–were used. Results obtained with each enzyme indicate strong similarities between the three nuclear envelope polypeptides. The amino acid compositions of the two most abundant polypeptides (P75 and P71) have been determined and found to be similar. Further, they readily yield large fragments upon brief alkaline hydrolysis. For both P75 and P71 the degree and the pattern of alkaline fragmentation are almost identical. A 61,000-dalton polypeptide which appears to be P61 is obtained from P75 and P71 by mild acid hydrolysis. These results establish the close chemical similarity of these predominant polypeptides in the erythrocyte nucleus and suggest that they serve related functions.     

Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS- polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000- 75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.     

Dimeric bisbenzimidazoles: Cytotoxicity and effects on DNA methylation in normal and cancer human cells

Cancer cells are characterized by hypermethylation of the promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors reactivate the genes, pointing to DNA methyltransferases as potential targets for anticancer therapy. Dimeric bisbenzimidazoles varying in the length of an oligomeric linker between two bisbenzimidazole residues (DB(n), where n is the number of methylene groups in the linker) were earlier shown to efficiently inhibit methylation of DNA duplexes by murine DNA methyltransferase Dnmt3a. Here, some of the compounds were tested for cytotoxicity, cell penetration, and effect on genomic DNA methylation in F-977 fetal lung fibroblasts and HeLa cervical cancer cells. Within the 0–60 μM concentration range, only DB(11) exerted a significant toxic effect on normal cells, whereas the effects of DB(n) on cancer cells were not significant. DB(1) and DB(3) slightly stimulated proliferation of HeLa and F-977 cells, respectively. DB(1) and DB(3) penetrated into the nuclei of HeLa and F-977 cells and accumulated predominantly in or near the nucleolus, while DB(11) was incapable of nuclear penetration. HeLa cells incubated with 26 μM DB(1) or DB(3) displayed a decrease in methylation of the 18S rRNA gene, which was in the regions of predominant accumulation of DB(1) and DB(3). The same DB(3) concentration exerted a similar effect on F-977 cells. However, the overall genomic DNA methylation level remained unchanged in both of the cell lines. The results indicated that DB(n)-type compounds can be used to demethylate certain genes and are thereby promising as potential anticancer agents.     

Analysis by Confocal Microscopy of the Behavior of Heat Shock Protein 70 within the Nucleus and of a Nuclear Matrix Polypeptide during Prolonged Heat Shock Response in HeLa Cells

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M_r = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42°C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.     

Carandinol: First isohopane triterpene from the leaves of Carissa carandas L. and its cytotoxicity against cancer cell lines

A new triterpene carandinol (1) was isolated from the leaves of Carissa carandas L., along with five known compounds, betulinic acid (2), β-sitosterol-3-O-β-d-glucopyranoside (3), oleanolic acid (4), ursolic acid (5) and 4-hydroxybenzoic acid (6). The structure of compound 1 was deduced as 3β,21α-dihydroxyisohopane by exhaustive spectroscopic analyses. The known compounds 2–6 were identified by comparison with the reported spectral data. Compound 1 was evaluated for cytotoxicity, immunomodulatory, antiglycation, antioxidant and enzyme inhibition activity. It exhibited significant in vitro cytotoxicity to every cell line tested (HeLa, PC-3 and 3T3) and was relatively more toxic to human cervical cancer (HeLa) cell line. This is the first report of the isolation of an isohopane triterpene from the genus Carissa. Carandinol also represents the first example of a cytotoxic isohopane.     

Cell type-specific differences in protein composition of nuclear pore complex-lamina structures in oocytes and erythrocytes of Xenopus laevis

Nuclear envelopes from oocytes of Xenopus laevis are rich in pore complexes and contain a major polypeptide of apparent molecular weight (M_r) 68,000. A rapid extraction procedure using buffer containing 1% ($\text{v}\text{v}$) Triton X-100 and 1.0 m-KCl allows the preparation of insoluble nuclear envelope skeletons showing only residual pore complex structures, with some interconnecting filament material, and one major polypeptide; i.e. that of M_r 68,000. This skeletal protein, which is not found in nuclear contents, reveals, on two-dimensional gel electrophoresis, a series of distinct isoelectric variants focusing in the pH range from 6.4 to 6.6. In living oocytes, this protein is continuously synthesized, as demonstrated by incorporation of labelled amino acids, and phosphorylated, A similar prominent skeletal protein has been found in nuclear envelopes of oocytes of other amphibia; however, slight but significant differences in electrophoretic mobility can be noted between different amphibian species.For comparison, nucleocortical lamina structures containing few pore complexes have been isolated, using similar extraction procedures, from various somatic cells of X. laevis, including erythrocytes. Laminae from these cells contain two major polypeptides, one (L_I) of M_r 72,000 focusing at approximately pH 5.35 and another (L_II) of M_r 69,000 focusing in several variants between pH 6.20 and 6.35. Similarly extracted “pore complex-lamina” fractions from rat liver contain a polypeptide of similar size and electrical charge as protein L_I from Xenopus and, in addition, two other polypeptides (M_r values: 74,000 and 62,000) both focusing between pH 6.6 and 6.9.It is concluded that the pore complex-lamina structure of the oocyte nucleus is assembled by only one major protein of M_r 68,000. The results also show that the protein composition of this insoluble nucleocortical structure can be different in different cells of the same organism. The compositional differences of these nuclear envelope skeletons are discussed in relation to the relative proportions of pore complex and interporous (lamina) material in the nuclear envelopes of the specific cells. It is suggested that the M_r 68,000 protein predominant in oocyte nuclear envelopes contributes, as an architectural component, to the formation of the highly organized nuclear pore complex.     

SINC,a type III secreted protein of Chlamydia psittaci,targets the inner nuclear membrane of infected cells and uninfected neighbors

SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci–infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP–transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear “lamina” structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci.     

Synthesis and cytotoxicity evaluation of highly functionalized pyranochromenes and pyranopyrans

A series of fluorinated tetrahydropyrano[3,2-c]chromenes and dihydropyrano[3,2-b]pyran derivatives have been synthesized and their in vitro cytotoxic activities have been determined in cervical cancer cell line (HeLa), human breast adenocarcinoma cell line (MDA-MB-231 and MCF-7) and human alveolar adenocarcinoma cell line (A549). Compounds 4g, 4k, 4p showed a very potent activity against MDA-MB-231, and 4c, 4p showed promising activity against MCF-7, while compounds 4c, 4g, 4p showed moderate activity against HeLa.     

Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)_n repeat] with high affinity and bind double-stranded telomeric DNA with a 100× reduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66–64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a λgt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.     

Thio-sugar motif of functional CARB-pharmacophore for antineoplastic activity. Part 2

Diverse functionalized representatives of (1-4)-S-thiodisaccharides, 6–9 were synthesized and assessed for cytotoxicity and apoptosis against human cancer cell lines (A549, LoVo, MCF-7 and HeLa). The FCP 6 was more active against MCF-7 cells (i.e., an estrogen-dependent breast cancer line), whereas other (1-4)-S-thiodisaccharides showed strongest activity against A549 cells (i.e., a lung adenocarcinoma line). We propose to use a concept of functional ‘CARB-pharmacophores’ when evaluating a potential for the compounds’ general antineoplastic activity. Future studies will determine the reasons for cell-type specificity of these compounds. The thio-sugar motif appears to be a promising lead for future developments.     

Induction of two transformation-sensitive membrane polypeptides in normal fibroblasts by a block in glycoprotein synthesis or glucose deprivation.

Following transformation of chick embryo fibroblasts (CEF) by avian RNA tumor viruses, two membrane polypeptides with apparent molecular weights of 90,000 and 75,000 daltons have been found to be increased (Stone, Smith and Joklik, 1974). We find that this alteration in membrane proteins is not directly related to transformation.The 90,000 and 75,000 dalton proteins are present in increased amounts in a 3T3 fibroblast mutant (AD6) defective in glycoprotein synthesis. Feeding the mutant N-acetylglucosamine, a metabolite that bypasses the metabolic block, restores the amount of these two proteins to the levels found in normal cells. The 75,000 dalton protein is markedly reduced, and the 90,000 dalton protein disappears and is replaced by a fully glycosylated derivative with a molecular weight of 92,000 daltons.Two glucose derivatives, glucosamine and 2-deoxyglucose, are known to interfere with the glycosylation process. The addition of these substances to normal CEF and 3T3 cells specifically induces the accumulation of the 90,000 and 75,000 dalton membrane polypeptides.Finally, the deprivation of glucose for 24–48 hr also induces the synthesis of the 90,000 and 75,000 dalton polypeptides in normal fibroblasts. The induction of these two proteins by glucose starvation suggests that they have a role in glucose utilization.     

Importance of nuclear localization for the apoptosis-induced activity of a fungal galectin AAL (Agrocybe aegerita lectin)

Agrocybe aegerita lectin (AAL) was identified previously in our group as a novel galectin from medicinal fungi Agrocybe aegerita, and has been shown to effectively induce cancer cell cycle arrest and apoptosis in vitro and tumor regression in vivo. Here, AAL was observed to translocate into the HeLa cell nucleus and induce cell apoptosis when it was predominantly in the nucleus. The N-terminus and C-terminus of AAL were required for nuclear localization. Site mutated proteins were generated based on AAL structure. Dimer interface mutant I25G, carbohydrate recognition domain (CRD) mutant R63H, and loop region mutant L33A could not enter the nucleus and lost the ability to induce apoptosis. CRD mutant H59Q and loop region mutant I144G maintained nuclear localization activity, and H59Q retained residual bioability but I144G had no activity, indicating that nuclear localization is important but not sufficient for AAL to become apoptotically active. Our findings provide a novel antitumor mechanism of fungal galectin.     

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.     

One-pot synthesis and biological evaluation of N-(aminosulfonyl)-4-podophyllotoxin carbamates as potential anticancer agents

A series of N-(aminosulfonyl)-4-podophyllotoxin carbamates were synthesized via the Burgess-type intermediate, and their antiproliferative activities were evaluated. Most of them possessed more potent cytotoxic effects against four human tumor cell lines (HeLa, A-549, HCT-8 and HepG2) and less toxic to normal human fetal lung fibroblast WI-38 cells than etoposide. In particular, N-(morpholinosulfonyl)-4-podophyllotoxin carbamate (9) exhibited the most potent activity towards these four tumor cells with IC₅₀ values in the range of 0.5–16.5 μM. Furthermore, immunofluorescence analysis revealed that 9 induced cell apoptosis by up-regulating the expression of p53 and ROS. Meanwhile, 9 effectively inhibited tubulin polymerization and microtubule assembly at cellular levels in HeLa cells. In addition, 9 could induce cell cycle arrest in the G2/M phase in HeLa cells by up-regulating levels of cyclinB1 and cdc2 and decreasing the expression of p-cdc2. These results indicated that 9 had potential for further development as anticancer agents.     

A potential CARB-pharmacophore for antineoplastic activity: Part 1

Diverse functionalized representatives of various classes of sugars, such as thio-, anhydro-, and sulfamido-sugars and myo-inositol oxide, were synthesized and assessed for cytotoxicity against human cancer cell lines (A549, LoVo, MCF-7 and HeLa). The inositol oxide (4) was more active against MCF-7 cells (i.e., an estrogen-dependent breast cancer line), whereas all 3 sulfur-containing compounds showed strongest activity against A549 cells (i.e., a lung adenocarcinoma line). We propose to use a concept of functional ‘CARB-pharmacophores’ when evaluating a potential for the compounds’ general antineoplastic activity. Future studies will determine the reasons for cell-type specificity of these compounds. The thio-sugar motif appears to be a promising lead for future developments.     

Synthesis of novel hetero ring fused pyridine derivatives; Their anticancer activity,CoMFA and CoMSIA studies

A series of novel furo[2,3-b]pyridine-2-carboxamide 4a–h/pyrido[3′,2′:4,5]furo[3,2-d] pyrimidin-4(3H)-one derivatives 5a–p were prepared from pyridin 2(1H) one 1 via selective O-alkylation with α-bromoethylester followed by cyclization, then reaction with different aliphatic primary amines to obtain 4 and further reaction with triethyl orthoacetate/triethyl orthoformate. Also prepared novel furo[2,3-b]pyridine-2-carbohydrazide Schiff’s bases 7a–h and pyrido [3′,2′:4,5]furo[3,2-d]pyrimidin-4(3H)-one derivatives 8a–h starting from furo[2,3-b]pyridine carboxylate derivatives 3 by reaction with hydrazine hydrate to form 6 and reaction with diverse substituted aldehydes and cyclization. Products 4a–h, 5a–p, 7a–h and 8a–h were screened against four human cancer cell lines (HeLa, COLO205, Hep G2 and MCF 7) and one normal cell line (HEK 293). Compounds 4e, 4f, 4g, 5h, 7c, 7d, 7e and 7f showed significant anticancer activity against all the cell lines at micro molar concentration and found to be non-toxic to normal cell line. Studies for HeLa, COLO205 and MCF-7 using CoMFA and CoMSIA. Models from 3D-QSAR provided a strong basis for future rational design of more active and selective HeLa, COLO205 and MCF-7 cell line inhibitors.     

Synthesis and biological activity of 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole derivatives,novel pro-apoptotic mitochondrial targeted agents

This study reports the synthesis of a number of 1- and 2-phenyl derivatives of the 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole nucleus, which were obtained by the reaction of the versatile 7-substituted 2,3-dihydro-3-hydroxymethylene-4H-1-benzothiopyran-4-ones with hydrazine and substituted phenylhydrazines. The antiproliferative activity of the synthesized compounds was evaluated by an in vitro assay on human tumor cell lines (HL-60 and HeLa) and showed a significant capacity of the 7-methoxy-substituted benzothiopyrano[4,3-c]pyrazoles 3b–d, carrying the pendant phenyl group in the 1-position, to inhibit cell growth. Investigation of the mechanism of action indicated the induction of the mitochondrial permeability transition (MPT) as the molecular event responsible for the inhibition of cell growth. This phenomenon is related to the ability of the test compounds to cause a rapid Ca²⁺-dependent and cyclosporin A-sensitive collapse of the transmembrane potential (ΔΨ) and matrix swelling. All this leads to the release of caspase activators, such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF), which trigger the pro-apoptotic pathway leading to DNA fragmentation.     

Cytotoxic diterpenoids as potential anticancer agents from the twigs of Casearia kurzii

A search for bioactive natural products as anticancer lead compounds has led to the isolation of five new clerodane diterpenoids (1–5) from the twigs of Casearia kurzii. Their structures were elucidated by extensive analysis of their NMR, IR, and HRESIMS data, and the absolute configurations were determined by experimental and calculated electronic circular dichroism (ECD) data analysis. The isolates were biologically evaluated and showed cytotoxic activities toward human lung cancer cells (A549), human cervical cancer cells (HeLa), and human hepatocellular carcinoma cells (HepG2). The most active compound (5) with an IC₅₀ value of 5.3 μM against HeLa cells, was found to induce apoptosis and arrest the HeLa cell cycle at G0/G1 stage to exert cytotoxic effects.     

Syntheses of 6-(2-hydroxy-6-phenylhexyl)-5,6-dihydro-2H-pyran-2-one derivatives and their cytotoxicities

The cytotoxicities against cancer cells (HL-60, HeLa) and insect cells (Sf9) of four stereoisomers of 6-(2-hydroxy-6-phenylhexyl)− 5,6-dihydro-2H-pyran-2-one (1) were evaluated, and then their structure-activity relationships examined. The 2′-dehydroxy derivative 5 of (6 R,2′R)- and (6 R,2′S)-1 showed the highest activity against HeLa cells (IC₅₀ = 1.4 μM). To evaluate the effect of the 2′-hydroxy group of 1, 6R-and 6S-oxetane derivatives were also synthesized and their activities examined. Against HeLa and HL-60 cells, the activities of the less potent stereoisomers were enhanced 3–4-fold by the introduction of the oxetane moieties at the 2′-position. Against the insect cell line (Sf9), phenyl derivative 7 showed the highest activity with an IC₅₀ value of 8.0 μM.     

Programmed cell death of secretory cavity cells of citrus fruits is associated with Ca2+ accumulation in the nucleus

Key message

An increase in Ca ²⁺ concentration in the nucleus may activate the PCD of secretory cavity cells, and further Ca ²⁺ accumulation contributes to the regulation of nuclear DNA degradation.

Abstract

Calcium plays an important role in plant programmed cell death (PCD). Previously, we confirmed that PCD was involved in the degradation of secretory cavity cells in Citrus sinensis (L.) Osbeck fruits. To further explore the function of calcium in the PCD of secretory cavity cells, we used potassium pyroantimonate precipitation to detect and locate calcium dynamics. At the precursor cell stage of the secretory cavity, Ca²⁺ was only distributed in the cell walls. At the early stage of secretory cavity initial cells, Ca²⁺ in the cell walls was gradually transported into the cytoplasm via pinocytotic vesicles. Although a small amount of Ca²⁺ was present in the nucleus, the TUNEL signal was scarcely observed. At the middle stage of initial cells, a large number of pinocytotic vesicles were transferred to the nucleus, where the vesicle membrane fused with the nuclear membrane to release calcium into the nucleoplasm. In addition, abundant Ca²⁺ aggregated in the condensed chromatin and nucleolus, where the TUNEL signal appeared the strongest. At the late stage of initial cells, the chromatin and nucleolus gradually degraded and disappeared, and the nucleus appeared broken-like, as Ca²⁺ in the cell wall had nearly completely disappeared, and Ca²⁺ in the nucleus was also rapidly reduced. Furthermore, the TUNEL signal also disappeared. These phenomena indicated that an increase in Ca²⁺ concentration in the nucleus might activate the PCD of secretory cavity cells, and further Ca²⁺ accumulation contributed to the regulation of nuclear DNA degradation.