Subtelomeric plasticity contributes to gene family expansion in the human parasitic flatworm Schistosoma mansoni.

BACKGROUND: The genomic region that lies between the telomere and chromosome body, termed the subtelomere, is heterochromatic, repeat-rich, and frequently undergoes rearrangement. Within this region, large-scale structural changes enable gene diversification, and, as such, large multicopy gene families are often found at the subtelomere. In some parasites, genes associated with proliferation, invasion, and survival are often found in these regions, where they benefit from the subtelomere's highly plastic, rapidly changing nature. The increasing availability of complete (or near complete) parasite genomes provides an opportunity to investigate these typically poorly defined and overlooked genomic regions and potentially reveal relevant gene families necessary for the parasite's lifestyle. RESULTS: Using the latest chromosome-scale genome assembly and hallmark repeat richness observed at chromosome termini, we have identified and characterised the subtelomeres of Schistosoma mansoni, a metazoan parasitic flatworm that infects over 250 million people worldwide. Approximately 12% of the S. mansoni genome is classified as subtelomeric, and, in line with other organisms, we find these regions to be gene-poor but rich in transposable elements. We find that S. mansoni subtelomeres have undergone extensive interchromosomal recombination and that these sites disproportionately contribute to the 2.3% of the genome derived from segmental duplications. This recombination has led to the expansion of subtelomeric gene clusters containing 103 genes, including the immunomodulatory annexins and other gene families with unknown roles. The largest of these is a 49-copy plexin domain-containing protein cluster, exclusively expressed in the tegument-the tissue located at the host-parasite physical interface-of intramolluscan life stages. CONCLUSIONS: We propose that subtelomeric regions act as a genomic playground for trial-and-error of gene duplication and subsequent divergence. Owing to the importance of subtelomeric genes in other parasites, gene families implicated in this subtelomeric expansion within S. mansoni warrant further characterisation for a potential role in parasitism.

Adenoviral vector-mediated expression of physiologic levels of human factor VIII in nonhuman primates.

An E1-, E2a-, E3-deleted adenoviral vector (Av3H82) encoding an epitope-tagged B domain-deleted human factor VIII cDNA (flagged FVIII) was evaluated in nonhuman primates. Twelve cynomolgus monkeys received intravenous administration of Av3H82; 6 monkeys received 6 x 10(11) particles/kg and another 6 received 3 x 10(12) particles/kg. Adenoviral vector transduction of the liver was efficient, reproducible, and linearly dose dependent. Physiologic levels of flagged FVIII were readily detected in plasma samples obtained from monkeys that received the higher dose of vector and human FVIII mRNA was detected in their livers. Expression of transgene mRNA was restricted to the liver by the albumin promoter. Although vector DNA was readily detected in the liver of monkeys that received the lower dose, neither human FVIII mRNA nor flagged FVIII protein could be detected. Vector distribution was widespread, with the highest levels observed in liver and spleen. Histopathology, hematology, and serum chemistry analysis identified the liver and blood as major sites of toxicity. Transient mild serum elevations of liver enzymes were observed, along with a dose-dependent inflammatory response in the liver. In addition, mild lymphoid hyperplasia was observed in the spleen. Mild anemia and a transient decrease in platelet count were observed, as was marrow hyperplasia and extramedullary hematopoiesis.

Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.

Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 0111:B4 LPS on movement of 14C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations > or = 0.5 ng/ml, and LPS (10 ng/ml) exposures of > or = 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 micrograms/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of > or = 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required.

Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: concurrent F-actin depolymerization and new actin synthesis.

Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: 1) transendothelial 14C-albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the DNAse 1 inhibition assay. LPS induced increments in 14C-albumin flux (P < 0.001) and intercellular gap formation at > or = 2-6 h. During this same time period the endothelial F-actin pool was not significantly changed compared to simultaneous media controls. Mean (+/- SE) G-actin (micrograms/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F-actin stabilization with phallicidin protected against the LPS-induced increments in G-actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS-induced decrement in F-actin (P = 0.0044), blunted the G-actin increment (P = 0.010), and increased LPS-induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS-induced F-actin depolymerization.

Effect of hypoxia and acidosis on the cytotoxicity of mitoxantrone, bisantrene and amsacrine and their platinum complexes at normal and hyperthermic temperatures.

In an effort to synthesize drugs which would become much more cytotoxic at clinically achievable hyperthermic temperatures, complexes of the tetrachloro-platinum(II) dianion were made with two anthracene dye derivatives, MITOX and BISANT, and the acridine dye derivative m-AMSA. As compared with the parent drug, PtCl4(MITOX)2 was less cytotoxic at 37 degrees C and more cytotoxic at 42 degrees C and 43 degrees C especially at pH 6.45. In contrast, the PtCl4(BISANT)2 was more cytotoxic than BISANT under all conditions. M-AMSA was again shown to be less cytotoxic at elevated temperatures but PtCl4(m-AMSA)2 was more cytotoxic especially at 43 degrees C and pH 6.45. Platinum levels in cells treated for 1 hr with 25 microM at 37 degrees C, 42 degrees C and at pH 7.40 versus pH 6.45 demonstrated no significant differences depending on temperature or pH except for PtCl4(MITOX)2 where approximately 4 times higher intracellular platinum levels were present at pH 6.45 versus pH 7.40, although this finding did not correlate with cytotoxicity. These results suggest that PtCl4(MITOX)2 and PtCl4(m-AMSA)2 may be highly interactive drugs with local hyperthermia.

Characteristics of five human tumor cell lines and sublines resistant to cis-diamminedichloroplatinum(II).

In order to study the mechanisms responsible for resistance to CDDP, 5 human tumor cell lines were made resistant to CDDP by repeated in vitro exposures. After cloning it was found that the cell lines developed were between 3.3-fold and 17-fold more resistant to CDDP than the parental cell lines at the IC90. These lines were also resistant to carboplatin and tetraplatin; however, resistance to tetraplatin was lower than to the other platinum complexes. Sensitivity was also assessed to Adria, MTX, 5-FU, chlorambucil, 4-HC, 4-HIF, BCNU, Thiotepa, HN2, Mito C and L-PAM, and no consistent cross-resistance was observed. As compared with the parental lines, non-protein sulfhydryl content was elevated in 3 resistant lines, and protein sulfhydryl was elevated in all 5 lines, as was glutathione-S-transferase activity. Measurements of platinum in whole cells and nuclei after exposure of the cultures to 25 microM CDDP for either 1 or 6 hr showed that nuclear levels reflected those in whole cells and that, per mg protein, platinum levels were lower in resistant cells at both time points. Formation of DNA cross-links, determined by alkaline elution, was lower in resistant cell lines than in parental cell lines, but did not correlate with the absolute cell kill observed. These results indicate that cellular resistance to CDDP often involves decreases in drug accumulation and increases in protein sulfhydryl content. Possible strategies for overcoming these mechanisms are discussed.

Influence of schedule on alkylating agent cytotoxicity in vitro and in vivo.

High dose, multiple alkylating agent chemotherapy is being employed in conjunction with autologous marrow transplantation in the clinic. We have investigated the scheduling of several alkylating drugs in an effort to optimize their antitumor effects. In vitro modeling of "continuous" (up to 72 h) versus "bolus" (1 h) exposure in MCF-7 cells showed that for N,N',N"-triethylenethiophosphoramide (thiotEPA), cis-diamminedichloroplatinum(II) (CDDP), 4-hydroperoxycyclophosphamide, carboplatin, and L-phenylalanine mustard (L-PAM) "continuous" exposure yielded essentially the same killing kinetics as "bolus" exposure. For N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), however, even with fresh drug additions every 30 min, "bolus" exposure produced superior cytotoxicity. In vivo modeling of "continuous" (three i.p. injections over 9 h) versus "bolus" (single dose) administration of the alkylating agents cyclophosphamide, BCNU, thiotEPA, melphalan, CDDP, and carboplatin was conducted in mice bearing EMT6 tumors, and tumor cell killing as measured by tumor cell survival in vitro was compared with killing of bone marrow (CFU-GM) measured in culture as a representative sensitive normal tissue. With cyclophosphamide there was a considerable increase in the therapeutic index (killing of tumor cells/killing of CFU-GMs) when the same total dose of drug was administered in multiple injections versus a single injection. For BCNU and thiotEPA, smaller increases in therapeutic index were observed. With L-PAM and CDDP, some advantage to multiple versus single dose administration was observed, and for carboplatin a decrease in the therapeutic index was seen. In conclusion, for all six alkylating agents examined, the multiple dose schedule was at least as effective against the tumor as the single dose schedule at all dose levels.

Effect of lonidamine on the cytotoxicity of four alkylating agents in vitro.

We examined the ability of lonidamine, which has been described as an inhibitor of cellular respiration and glycolysis, to enhance the cytotoxicity of alkylating agents to MCF-7 human breast-carcinoma cells. Lonidamine was increasingly cytotoxic to MCF-7 cells with increasing time of exposure. With a 12-h exposure, the IC50 for lonidamine was about 365 microM, and with a 24-h exposure it was about 170 microM. A drug concentration of 250 microM was chosen for use in the drug combination studies. Lonidamine appeared to have a dose-modifying effect on cisplatin (CDDP), producing increasingly supra-additive cell kill with increasing CDDP concentration. When simultaneously incubated with lonidamine for 1 h, 500 microM CDDP yielded a cell kill that was 2 log greater than additive cytotoxicity. Extending the exposure to lonidamine for 12 h after CDDP treatment led to a small, additional aliquot of cell kill of about 2.5-fold over the CDDP concentration range. Lonidamine also appeared to have a dose-modifying effect on melphalan cytotoxicity in the melphalan concentration range of 100-500 microM. Between concentrations of 10 and 100 microM melphalan, the drug combination survival after 1 h exposure fell within the envelope of additivity for the two agents. However, maintaining the presence of lonidamine for an additional 12 h increased the effect such that the combination was supra-additive over the entire concentration range of melphalan. Simultaneous exposure to 4-hydroperoxycyclophosphamide (4-HC) and lonidamine for 1 h resulted in greater than additive cell kill, and extending the lonidamine exposure period such that lonidamine was present during and 12 h after 4-HC treatment further increased this effect. Lonidamine had a moderate effect on the cytotoxicity of carmustine (BCNU) with a 1 h simultaneous exposure; however, this treatment combination reached greater than additive cytotoxicity only at the highest concentration of BCNU tested. Extending the lonidamine exposure time for an additional 12 h resulted in supra-additive cell kill over the BCNU concentration range. Therefore, when lonidamine was present during exposure to the alkylating agent and its presence was then extended for an additional 12 h, a synergistic cell kill was produced with all four alkylating agents tested.