Synthesis and in vitro properties of trimethylamine- and phosphonate-substituted carboranylporphyrins for application in BNCT

A series of carboranylporphyrins containing either amine or phosphonic acid functionalities and two to six closo-carborane clusters have been synthesized via a [2+2] condensation of a dimethylamino- or diethylphosphonate-substituted dipyrromethane with a dicarboranylmethyl-benzaldehyde. The X-ray structures of four key reaction intermediates (1, 2, 3, and 4a) and of two target porphyrins, the diphosphonate ester- and the diamino-tetracarboranylporphyrins 5b and 6a, are presented and discussed. In vitro studies using human carcinoma HEp2 and human glioblastoma T98G cells show that these porphyrins are non-toxic in the dark up to 100 microM concentrations, and that a tetracarboranylporphyrin bearing two quaternary ammonium groups is the most efficiently taken up by cells at short times (up to 8 h), followed by a dicarboranylporphyrin bearing three phosphonic acid substituents. All carboranylporphyrins delivered therapeutic amounts of boron to T98G cells and localized mainly within the cell lysosomes.     

Synthesis and cellular studies of a carboranylchlorin for the PDT and BNCT of tumors

The syntheses of closo- and nido-carboranylchlorins 4 and 5 from a known carboranylporphyrin are described. Water-soluble nido-carboranylporphyrin 5 was found to have very low dark cytotoxicity (IC50 > 500 microM using a MTT-based assay) but to be toxic in the presence of red light (IC50 = 80 microM at 0.55 J/cm2 light dose). Under the same experimental conditions, carboranylchlorin 5 was taken up by human glioma T98G cells to a significantly higher extent than chlorin e6, a chlorophyll degradation product. The preferred sites of subcellular localization of carboranylchlorin 5 were found to be the cell lysosomes. Our results suggest that carboranylchlorin 5 is a promising new dual sensitizer for the PDT and BNCT treatment of tumors.     

Selective decrease of SN1(SNAT3) mRNA expression in human and rat glioma cells adapted to grow in acidic medium

The system N glutamine (Gln) transporter SN1(SNAT3) is overexpressed in human malignant glioma cells in situ as compared to the adjacent brain tissue or metastases from different organs [Sidoryk, M., Matyja, E., Dybel, A., Zielińska, M., Bogucki, J., Jaskólski, D.J., Liberski, P.P., Kowalczyk, P., Albrecht, J., 2004]. Increased expression of a glutamine transporter SNAT3 is a marker of malignant gliomas. NeuroReport 15, 575-578], but its role in tumor growth as compared to the other Gln transporters is unknown. One of the profound, growth-promoting effects of glial tumor in situ is acidification of the extracellular space. In the kidney SN1(SNAT3) mRNA participates in the adaptation to acidosis. In this study therefore, expression of mRNAs coding for SN1(SNAT3) and other Gln transporters was measured in human (T98G) and rat (C6) glioma cells incubated for 4h in an acidic medium (AI) (pH 6.5). MTT assay revealed no cell loss in AI cells, and intracellular pH (pHi) as measured by a fluorescent probe (BCECF-AM) was slightly alkaline in C6 and T98G cells, indicating that the cells have adapted to AI. AI significantly decreased the SN1(SNAT3) mRNA expression in C6 (a 60% decrease) and T98G cells (a 50% decrease). The decrease retreated in C6 cells 4h after transferring them back to the neutral medium. The expression of ASCT2 mRNA (system ASC), ATA1 mRNA (system A) and SN2(SNAT5) mRNA (system N) were not affected by AI in either of the cell lines. [(3)H]Gln uptake in C6 or T98G cells grown in neutral medium was mainly mediated by system ASCT2: system N contributed to only approximately 7% of the uptake. AI did not affect the total Gln uptake, and only slightly decreased the system N-mediated component of the uptake. Hence, SN1(SNAT3) does not seem to be involved in the adaptation of cultured glioma cells to acidic millieu.     

Secretion of metabolites of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one by Hep G2 cells

We demonstrated that two-thirds of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one was efficiently taken up and consequently metabolized and secreted by Hep G2 cells when it was pulsed for 16 hrs followed by chasing for another 72 hrs. The metabolism was clearly reflected by the cellular secretion. Approximately 61%, 26% and 10% of uptaken [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one was metabolized to its water-soluble metabolites, polar metabolites in lipid phase and ketosteryl esters, respectively. Ninety-four percent of these metabolites was secreted into media. Interestingly, polar forms of the metabolites of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one accounted for over 95% of the cellular secretes. Limited secretion of ketosteryl esters was also detected. The data strongly suggest that Hep G2 cells have the potential to process 5 alpha-cholest-8(14)-en-3 beta-ol-15-one and could provide a good model for studying its secretion.     

Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. II. Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of Gi-coupled signalling

Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301-19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/G alpha o1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/G alpha o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein alpha subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/G alpha o1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells.     

Antibody epitopes on the neuraminidase of a recent H3N2 influenza virus (A/Memphis/31/98)

We have characterized monoclonal antibodies raised against the neuraminidase (NA) of a Sydney-like influenza virus (A/Memphis/31/98, H3N2) in a reassortant virus A/NWS/33(HA)-A/Mem/31/98(NA) (H1N2) and nine escape mutants selected by these monoclonal antibodies. Five of the antibodies use the same heavy chain VDJ genes and may not be independent. Another antibody, Mem5, uses the same V(H) and J genes with a different D gene and different isotype. Sequence changes in escape mutants selected by these antibodies occur in two loops of the NA, at amino acid 198, 199, 220, or 221. These amino acids are located on the opposite side of the NA monomer to the major epitopes found in N9 and early N2 NAs. Escape mutants with a change at 198 have reduced NA activity compared to the wild-type virus. Asp198 points toward the substrate binding pocket, and we had previously found that a site-directed mutation of this amino acid resulted in a loss of enzyme activity (M. R. Lentz, R. G. Webster, and G. M. Air, Biochemistry 26:5351-5358, 1987). Mutations at residue 199, 220, or 221 did not alter the NA activity significantly compared to that of wild-type NA. A 3.5-A structure of Mem5 Fab complexed with the Mem/98 NA shows that the Mem5 antibody binds at the sites of escape mutation selected by the other antibodies.     

Nucleoside analogue substitutions in the trinucleotide DNA template recognition sequence 3'-(CTG)-5' and their effects on the activity of bacteriophage T7 primase

Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d[CTGG(G/T)]-5' and 3'-(CTGTG)-5', respectively. The 3'-terminal deoxycytidine residue is conserved but noncoding. A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined. The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described. Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used for a critical interaction by the enzyme. Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H, 3H]-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue. Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis.     

N⁴-Phenyl-substituted 2-acetylpyridine thiosemicarbazones: cytotoxicity against human tumor cells, structure-activity relationship studies and investigation on the mechanism of action

N(4)-Phenyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-(1-(pyridin-2-yl)ethylidene)hydrazinecarbothioamide) and its N(4)-ortho-, -meta- and -para-fluorophenyl (H2Ac4oFPh, H2Ac4mFPh, H2Ac4pFPh), N(4)-ortho-, -meta- and -para-chlorophenyl (H2Ac4oClPh, H2Ac4mClPh, H2Ac4pClPh), N(4)-ortho-, -meta- and -para-iodophenyl (H2Ac4oIPh, H2Ac4mIPh, H2Ac4pIPh) and N(4)-ortho-, -meta- and -para-nitrophenyl (H2Ac4oNO(2)Ph, H2Ac4mNO(2)Ph, H2Ac4pNO(2)Ph) derivatives were assayed for their cytotoxicity against human malignant breast (MCF-7) and glioma (T98G and U87) cells. The compounds were highly cytotoxic against the three cell lineages (IC(50): MCF-7, 52-0.16 nM; T98G, 140-1.0 nM; U87, 160-1.4 nM). All tested thiosemicarbazones were more cytotoxic than etoposide and did not present any haemolytic activity at up to 10(-5)M. The compounds were able to induce programmed cell death. H2Ac4pClPh partially inhibited tubulin assembly at high concentrations and induced cellular microtubule disorganization.     

Formation and characterization of three dimensional human hepatocyte cell line spheroids on chitosan matrix for in vitro tissue engineering applications

Chitosan was used as a matrix to induce three-dimensional spheroids of HepG2 cells. Chitosan films were prepared and used for culturing Hep G2 cells. Attachment kinetics of the cells was studied on the chitosan films. The optimum seeding density of the Hep G2 cells, required for three-dimensional spheroid formation was determined and was found to be 5 × 10⁴/ml. The growth kinetics of Hep G2 cells was studied using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay, and morphology of the cells was studied through optical photographs taken at various days of culture. The liver cell functions of the spheroids were determined by measuring albumin and urea secretions. The results obtained from these studies have shown that the culture of Hep G2 cells on chitosan matrix taking appropriate seeding density resulted in the formation of three-dimensional spheroids and exhibited higher amount of albumin and urea synthesis compared to monolayer culture. These miniature “liver tissue like” models can be used for in vitro tissue engineering applications like preliminary evaluation of the toxicity of drugs and chemicals.     

Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is discussed.     

Porphyrin-retinamides: synthesis and cellular studies

A series of four porphyrin-retinamides containing either all-trans- or 13-cis-retinoid acid residues, directly linked to the para-phenyl position of meso-tetraphenylporphyrin or via a low-molecular-weight PEG spacer, have been synthesized. The biological properties of these conjugates were evaluated in a model cell line, human HEp2, and in neuroblastoma SK-N-DZ cells, which exhibit moderate expression of retinoic acid receptors and retinoic acid-induced differentiation. The directly linked porphyrin-retinamides were taken up by a greater extent (20-50% more) in SK-N-DZ than in HEp2 cells. However, the PEG-containing conjugates accumulated maximally within both cell lines and approximately by the same amount, probably due to their increased amphiphilicity. Among all conjugates, the porphyrin-PEG-13-cis-retinamide accumulated the most in both cell lines (about 5 times more than the non-pegylated conjugates). None of the porphyrin-retinamide conjugates were toxic toward HEp2 cells at concentrations up to 100 microM, and only the hydrophobic non-pegylated conjugates were moderately toxic to SK-N-DZ cells [IC50 (dark) = 56-92 microM, and IC50 (at 1 J/cm2) = 6-8 microM]. All conjugates preferentially localized within cellular vesicles that correlated well to the lysosomes and, in addition, the PEG-containing porphyrin-retinamides were also found in the ER.     

Synthesis, cellular uptake and animal toxicity of a tetra(carboranylphenyl)-tetrabenzoporphyrin

A water-soluble nido-carboranyl-tetrabenzoporphyrin has been synthesized in 43% overall yield, by condensation of butanopyrrole with a carboranylbenzaldehyde, followed by metal insertion, oxidation, demetallation and deboronation reactions. This compound accumulated within human glioblastoma T98G cells to a significant higher extent than a structurally related nido-carboranylporphyrin, and localized preferentially in the cell lysosomes. Animal toxicity studies using male and female BALB/c mice revealed that both compounds are non-toxic even at a dose of 160 mg/kg, administered intraperitoneally as a single injection at a concentration of 4 mg/mL. It is concluded that the tetra(carboranylphenyl)-tetrabenzoporphyrin is a promising new sensitizer for the treatment of malignant tumors.     

Quantitative analysis of glioma cell invasion by confocal laser scanning microscopy in a novel brain slice model

To quantitatively analyze the spatial extent of glioma cell migration in an organotypic brain slice culture, we developed a new invasion model with the aid of confocal laser scanning microscopy (CLSM). CLSM allowed not only for three-dimensional visualization of the invasive pattern of human T98G glioma cells in the living brain slice but also for serial analysis of the invasive process over several weeks. Twenty-four hours after the T98G glioma spheroid was initiated to coculture with a brain slice, the glioma cells detached themselves from the spheroid and spontaneously continued to migrate on the surface of the brain slice, while they diffusely invaded into the slice by migrating to a deeper site. Immunohistochemical analysis revealed that these migrating glioma cells much more strongly immunostained for matrix metalloproteinase (MMP)-2 and -9 than the tumor spheroid which remained at the implanted site. Treatment of the T98G glioma spheroid with 1,10-phenanthroline, a specific inhibitor of MMPs, significantly inhibited not only the cell migration on the surface of the brain slice but also the invasion of the glioma cells into the slice. The present version of the glioma invasion model using CLSM makes it possible to spatially and serially analyze the extent of glioma cell invasion in the living brain slice for several weeks, making it a very useful tool for investigating the cellular and molecular mechanisms of glioma invasion under conditions most analogous to those of normal brains in vivo.     

In vitro toxicity of palladium(II) and gold(III) porphyrins and their aqueous metal ion counterparts on Trypanosoma brucei brucei growth

The trypanocidal effects of aqueous gold(III) and palladium(II) and their metalloporphyrin derivatives on Trypanosoma brucei brucei growth in culture have been studied using an Alamar Blue indicator assay. All the experiments were conducted in the dark. As previously described for mercury(II), cadmium(II) and lead(II) porphyrins [Chem.-Biol. Interact. 139 (2002) 177], the toxicity of the metalloporphyrin complex of palladium(II) to T. b. brucei parasites was much higher compared to the aqueous free palladium(II) and free base porphyrin. Palladium(II) porphyrin, free palladium(II), and the free base porphyrin were trypanocidal to T. b. brucei at concentrations >1.5 x 10(-6), >6.1 x 10(-6) and >1.9 x 10(-5) M, respectively. While gold(III) porphyrin was effective against the parasites at concentrations >4.8 x 10(-6) M, its aqueous gold(III) was toxic at concentrations as low as 2.0 x 10(-7) M due to the generation of free radicals in the presence of this metal ion which enhanced its toxicity to the T. b. brucei parasites. Although some cell division was observed in some of the cells treated with palladium(II) porphyrin, some dividing cells had no nucleus due to unequal division and delivery of the nuclei into the daughter cells. As a result, the rate of cell division decreased with time and cell death occurred within 24 h. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. Of all the porphyrins and free metal ions tested, only mercury(II) porphyrin and aqueous gold(III) ion were toxic to the trypanosomes in the 10(-7) M range. The chemotherapeutic potential of these observations is discussed.     

Mechanisms of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (Dacarbazine) cytotoxicity toward Chinese hamster ovary cells in vitro are dictated by incubation conditions

Decomposition of the antitumor agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, Dacarbazine) produces several potentially toxic compounds, the concentration of which depend on incubation parameters such as pH, temperature and illumination. The action of DTIC on chinese hamster ovary (CHO) cell clone formation in the dark (7-8-day incubation) reflects the slow formation of 2-azahypoxanthine. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8)-deficient cells are resistant to DTIC under these conditions, reflecting their inability to utilize 2-azahypoxanthine. The toxicity of DTIC in conventional survival experiments (1-2-h exposure to drug) is dependent upon illumination and is highly influenced by the pH of the medium. Toxicity of DTIC in these experiments appears to reflect rapid accumulation of the immediate photodecomposition product of the drug, 4-diazoimidazole-5-carboxamide (DZC), since HGPRT-deficient cells are not resistant to DTIC under these conditions. The biologically initiated pathway of DTIC action (enzymatic hydroxylation) has little, if any, role in the action of this agent toward cultured CHO cells.     

Arsenic trioxide induces different gene expression profiles of genes related to growth and apoptosis in glioma cells dependent on the p53 status

We have previously reported that As(2)O(3) affected cell cycle progression and cyclins D1 and B1 expression in two glioma cell lines differing in p53 status (U87MG-wt; T98G-mutated). In the present study, we further demonstrated that As(2)O(3) affected proliferation, viability and apoptosis of the two cell lines in a dose- and time-dependent manner, and T98G cells were more sensitive than U87MG cells to As(2)O(3) -induced apoptosis and inhibition of proliferation and viability. We further investigated the expression profiles of genes related with apoptosis and cell cycle in the two cell lines with a human cDNA-microarray (SuperArray) spotted with 267 genes of apoptosis and cell cycle. Thirty five genes were upregulated and 15 genes downregulated at least 2-fold by As(2)O(3) in U87-MG cells; whereas, 38 genes were upregulated and 21 genes downregulated at least 2-fold in T98G cells by As(2)O(3). After As(2)O(3) treatment, p53 expression was upregulated 56.5-fold in T98G cells, but only 6.0-fold in U87MG cells. The results indicate that As(2)O(3) suppresses the growth of U87MG cells mainly by regulating expression of genes of cell cycle arrest, stress and toxicity; whereas As(2)O(3) affects T98G cells mainly by regulating expression of genes belonging to Bcl-2, tumor necrotic factor receptor and ligand families. The data may be helpful for optimizing As(2)O(3) as an anti-cancer drug in the treatment of gliomas.     

Short-term effect on intestinal epithelial Na(+)/H(+) exchanger by Gi(alpha1,2)-coupled 5-HT(1A) and G(q/11)-coupled 5-HT(2) receptors

The present study evaluated the effect of 5-hydroxytryptamine (5-HT) on intestinal Na(+)/H(+) exchanger (NHE) activity and the cellular signaling pathways involved in T84 cells. T84 cells express endogenous NHE1 and NHE2 proteins, detected by immunoblotting, but not NHE3. The rank order for inhibition of NHE activity in acid-loaded T84 cells was 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; IC(50)=519 [465, 579] nM)>cariporide (IC(50)=630 [484, 819] nM)>amiloride (IC(50)=19 [16, 24] microM); the NHE3 inhibitor S3226 was found to be devoid of effect. This different inhibitory sensitivity indicates that both NHE1 and NHE2 isoforms may play an active role in Na(+)-dependent intracellular pH (pH(i)) recovery in T84 cells. Short-term exposure (0.5 h) of T84 cells to 5-HT increased NHE activity in a concentration-dependent manner. The stimulation induced by 5-HT (30 microM) was partially inhibited by both WAY 100135 (300 nM) and ketanserin (300 nM), antagonists of 5-HT(1A) and 5-HT(2) receptors, respectively. NHE activity was significantly increased by 8-OH-DPAT and alpha-methyl-5-HT, agonists of, respectively, 5-HT(1A) and 5-HT(2) receptors. An incubation of T84 cells with anti-G(s) and anti-G(beta) antibodies complexed with lipofectin did not prevent the 5-HT-induced stimulation of NHE activity. Overnight treatment with anti-G(ialpha1,2) and anti-G(q/11) antibodies complexed with lipofectin blocked the stimulatory effect induced by 8-OH-DPAT and alpha-methyl-5-HT, respectively. It is concluded that in T84 cells 5-HT enhances intestinal NHE activity through stimulation of G(ialpha1,2)-coupled 5-HT(1A) and G(q/11)-coupled 5-HT(2) receptors.     

Time-dependent changes in H1 content, H1 turnover, DNA elongation, and the survival of cells blocked in early S phase by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine

Cells were synchronized in G1 by isoleucine deprivation and then released into medium containing 1 mM hydroxyurea (HU), 5 micrograms mL-1 aphidicolin (APC), or 1 microgram mL-1 5-fluorodeoxyuridine (fl5dU). Coulter volume, content of histone H1 per unit DNA, turnover of histone H1, the extent of DNA elongation, and the survival of cells were measured as functions of time after release into the presence of the drugs. At the concentrations used in the experiments, the drug differ in their toxicity (fl5dU greater than HU greater than APC), induction of unbalanced cell growth, and the distribution of new DNA fragment sizes allowed during block, but they all (1) allow cells to enter S phase, (2) cause similar time-dependent losses of histone H1 per unit DNA, which begin as synchronized G1 cells begin to enter S phase, (3) retard DNA elongation beyond replicon size, and (4) retard the turnover of histone H1. The results indicate that loss of histone H1, inhibition of histone turnover, the retarded ligation of newly replicated DNA into bulk chromatin, and chromatin structural changes may be part of the cell's general response to inhibition of DNA replication. Since transient S phase block increases the frequencies of gene amplification [Mariani, B. D., & Schimke, R. T. (1984) J. Biol. Chem. 259, 1901-1910] and sister chromatid exchanges (SCE) [Rainaldi, G., Sessa, M. R., & Mariani, T. (1984) Chromosoma 90, 46-49], the observed changes in H1 content and chromatin organization may also be essential features of gene amplification and SCE.     

Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells

Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been assumed to be as a result of the extensive accumulation of beta-amyloid (Abeta). In addition to Abeta fibrillar assemblies, there are pre-fibrillar forms that have been shown to be neurotoxic, although their role in cholinergic degeneration is still not known. Using the cholinergic cell line SN56.B5.G4, we investigated the effect of different Abeta(1-42) aggregates on cell viability. In our model, only soluble oligomeric but not fibrillar Abeta(1-42) forms induced toxicity in cholinergic cells. To determine whether the neurotoxicity of oligomeric Abeta(1-42) was caused by its oxidative potential, we performed microarray analysis of SN56.B5.G4 cells treated either with oligomeric Abeta(1-42) or H(2)O(2). We showed that genes affected by Abeta(1-42) differed from those affected by non-specific oxidative stress. Many of the genes affected by Abeta(1-42) were present in the endoplasmic reticulum (ER), Golgi apparatus and/or otherwise involved in protein modification and degradation (chaperones, ATF6), indicating a possible role for ER-mediated stress in Abeta-mediated toxicity. Moreover, a number of genes, which are known to be involved in AD (clusterin, Slc18a3), were identified. This study provides important leads for the understanding of oligomeric Abeta(1-42) toxicity in cholinergic cells, which may account in part for cholinergic degeneration in AD.     

Metabolism and selectivity of arabinonucleoside in human lymphoid cells

The selective toxicity of purine deoxynucleosides against lymphoid cells appears to be mediated by a preferential accumulation of the corresponding triphosphates in these cells. We report a study of the metabolism and toxicity of arabinonucleosides of guanine and cytosine toward human T- and B-lymphoblastoid-cell lines. Both compounds inhibited the growth of T lymphoblasts at concentrations less than 2 microM. However, only ara-G exhibited a strong selectivity for T lymphocytes as indicated by a 100-fold greater toxicity to T than B cells. ara-G is not significantly degraded to guanine but is metabolized to the triphosphate. In common with the other arabinonucleoside, cytotoxicity by ara-G was associated with specific inhibition of DNA synthesis in cells. The capacity of T cells (CCRF-CEM) to accumulate ara-GTP was dependent primarily on deoxycytidine kinase. The level of intracellular ara-GTP accumulated after incubation with the corresponding nucleoside was 20- to 40-fold higher in T cells than either of two B-lymphoblast-cell lines, WI-L2 or PF-2S. The levels of phosphorylating activity for ara-C in extracts of T- and B-cell lines were approximately equal; in contrast, ara-G phosphorylating activity was four- to fivefold higher in B lymphoblasts. After removal of arabinonucleosides from the culture medium, ara-GTP levels in B lymphoblasts declined at a rate that was two to four times faster than that of ara-CTP. In marked contrast, no catabolism of the arabinonucleoside triphosphates was detected in T lymphoblasts. These results suggest that the selectivity of arabinonucleosides to human lymphoid cells of various phenotypes can be correlated with their nucleotide metabolism. The selectivity of ara-G for T and B cells can be correlated with their differential ability to catabolize ara-GTP.