Fhit-deficient normal and cancer cells are mitomycin C and UVC resistant.

To identify functions of the fragile tumour suppressor gene, FHIT, matched pairs of Fhit-negative and -positive human cancer cell clones, and normal cell lines established from Fhit -/- and +/+ mice, were stressed and examined for differences in cell cycle kinetics and survival. A larger fraction of Fhit-negative human cancer cells and murine kidney cells survived treatment with mitomycin C or UVC light compared to matched Fhit-positive cells; approximately 10-fold more colonies of Fhit-deficient cells survived high UVC doses in clonigenic assays. The human cancer cells were synchronised in G1, released into S and treated with UVC or mitomycin C. At 18 h post mitomycin C treatment approximately 6-fold more Fhit-positive than -negative cells had died, and 18 h post UVC treatment 3.5-fold more Fhit-positive cells were dead. Similar results were obtained for the murine -/- cells. After low UVC doses, the rate of DNA synthesis in -/- cells decreased more rapidly and steeply than in +/+ cells, although the Atr-Chk1 pathway appeared intact in both cell types. UVC surviving Fhit -/- cells appear transformed and exhibit >5-fold increased mutation frequency. This increased mutation burden could explain the susceptibility of Fhit-deficient cells in vivo to malignant transformation.

Cytotoxic sesquiterpenoids from Ratibida columnifera.

Bioassay-directed fractionation of the flowers and leaves of Ratibida columnifera using a hormone-dependent human prostate (LNCaP) cancer cell line led to the isolation of 10 cytotoxic substances, composed of five novel xanthanolide derivatives (2-4, 7, and 8), a novel nerolidol derivative (9), and three known sesquiterpene lactones, 9alpha-hydroxy-seco-ratiferolide-5alpha-O-angelate+ ++ (1), 9alpha-hydroxy-seco-ratiferolide-5alpha-O-(2-methylbut yrate) (5), 9-oxo-seco-ratiferolide-5alpha-O-(2-methylbutyrate) (6), as well as a known flavonoid, hispidulin (10). On the basis of its cytotoxicity profile, compound 5 was selected for further biological evaluation, and was found to induce G1 arrest and slow S traverse time in parental wild type p53 A2780S cells, but only G2/M arrest in p53 mutant A2780R cells, with strong apoptosis shown for both cell lines. The activity of 5 was not mediated by the multidrug resistance (MDR) pump, and it was not active against several anticancer molecular targets (i.e., tubulin polymerization/depolymerization, topoisomerases, and DNA intercalation). While these results indicate that compound 5 acts as a cytotoxic agent via a novel mechanism, this substance was inactive in in vivo evaluations using the murine lung carcinoma (M109) and human colon carcinoma (HCT116) models.

Synthesis and biological evaluation of analogues of cryptolepine, an alkaloid isolated from the Suriname rainforest.

Bioassay-guided fractionation of an extract of a mixture of Microphilis guyanensis and Genipa americanacollected in the rainforest of Suriname yielded the known alkaloid cryptolepine (2) as the major active compound in a yeast bioassay for potential DNA-damaging agents; the same compound was later reisolated from M. guyanensis. The structure of cryptolepine was identified unambiguously by spectral data and by its total synthesis. Several cryptolepine derivatives (3-29, 32-41) were synthesized based on modifications of the C-2, N-5, N-10, and C-11 positions. Two cryptolepine dimers (30, 31) were also prepared. The structure modifications did not result in compounds with a higher potency than the parent compound cryptolepine in the yeast assay system, although some derivatives did show significant activity. Selected compounds (6, 7, 17, 22, 23, 26, and 27) were also tested for cytotoxicity in mammalian cell culture, and two compounds showed significant cytotoxic activity.

Chronic inflammation and susceptibility to bacterial infections in mice lacking the polypeptide (p)105 precursor (NF-kappaB1) but expressing p50.

The polypeptide (p)50 molecule, a subunit of nuclear factor (NF)-kappaB, is produced after proteolytic processing of the p105 precursor (NF-kappaB1). Although the p105 precursor has been postulated to play a role in the regulation of the Rel/NF-kappaB activity, its physiological relevance remains unclear. To investigate that, we generated mutant mice lacking the COOH terminal half of the p105 precursor, but expressing the p50 product (p105-/-). These mutant mice displayed an inflammatory phenotype composed of lymphocytic infiltration in lungs and liver, and an increased susceptibility to opportunistic infections. Enlargement of multiple lymph nodes, splenomegaly due to erythrocytic extramedullary hematopoiesis, and lymphoid hyperplasia were also observed in p105-/- mice. Cytokine production in p105-/- macrophages was severely impaired, whereas proliferative responses of p105-/- B cells were increased. T cell functions were only moderately impaired in mutant mice. Loss of p105 also led to enhanced constitutive p50 homodimer and inducible NF-kappaB activities in unstimulated and stimulated cells, respectively. As several genes regulated by Rel/NF-kappaB were upregulated in p105-/- thymus but downregulated in p105-/- macrophages, the enhanced p50 homodimers appear to function as transcriptional activators or repressors, depending on the cell type. Thus, the p105 precursor is indispensable in the control of p50 activity, and lack of the precursor has distinct effects on different cells.

Nuclear factor (NF)-kappa B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses.

The nfkb2 gene is a member of the Rel/NF-kappa B family of transcription factors. COOH-terminal deletions and rearrangements of this gene have been associated with the development of human cutaneous T cell lymphomas, chronic lymphocytic leukemias, and multiple myelomas. To further investigate the function of NF-kappa B2, we have generated mutant mice carrying a germline mutation of the nfkb2 gene by homologous recombination. NF-kappa B2-deficient mice showed a marked reduction in the B cell compartment in spleen, bone marrow, and lymph nodes. Moreover, spleen and lymph nodes of mutant mice presented an altered architecture, characterized by diffuse, irregular B cell areas and the absence of discrete perifollicular marginal and mantle zones; the formation of secondary germinal centers in spleen was also impaired. Proliferation of NF-kappa B2-deficient B cells was moderately reduced in response to lipopolysaccharide, anti-IgD-dextran, and CD40, but maturation and immunoglobulin switching were normal. However, nfkb2 (-/-) animals presented a deficient immunological response to T cell-dependent and -independent antigens. These findings indicate an important role of NF-kappa B2 in the maintenance of the peripheral B cell population, humoral responses, and normal spleen architecture.

Expression of constitutively active IkappaB beta in T cells of transgenic mice: persistent NF-kappaB activity is required for T-cell immune responses.

The transcription factor NF-kappaB is normally sequestered in the cytoplasm by members of the IkappaB family, including IkappaB alpha, IkappaB beta, and the recently cloned IkappaB epsilon. Upon cellular activation, these inhibitors are rapidly phosphorylated on two amino-terminal serines, ubiquitinated, and degraded by the 26S proteasome, releasing a functional NF-kappaB. To determine the importance of IkappaB beta in NF-kappaB regulation in T cells, we generated transgenic mice expressing a constitutively active IkappaB beta mutant (mIkappaB beta) under the control of the lck promoter. The transgene contains the two critical N-terminal serine residues mutated to alanines and therefore no longer susceptible to degradation upon cell activation. mIkappaB beta is unable to totally displace IkappaB alpha from RelA-containing complexes, thus allowing a transient activation of NF-kappaB upon T-cell stimulation. However, mIkappaB beta completely blocks NF-kappaB activity after IkappaB alpha degradation. In addition, as a consequence of this inhibition, ikba expression is down regulated, along with that of other NF-kappaB-regulated genes. These transgenic mice have a significant reduction in the peripheral T-cell population, especially CD8+ cells. The remaining T cells have impaired proliferation in response to phorbol 12-myristate 13-acetate plus phytohemagglutinin or calcium ionophore but not to anti-CD3/anti-CD28 costimulation. As a result of these alterations, transgenic animals present defects in immune responses such as delayed-type hypersensitivity and the generation of specific antibodies against T-cell-dependent antigens. These results show that in nonstimulated T cells, IkappaB beta cannot efficiently displace IkappaB alpha bound to RelA-containing complexes and that persistent NF-kappaB activity is required for proper T-cell responses in vivo.

Studies on the effect of CL 306,293, a substituted quinoline carboxylic acid, on the clinical disease induced in mice with LP-BM5 virus.

CL 306,293, a substituted quinoline carboxylic acid, is a potent inhibitor of dihydroorotic acid dehydrogenase, an enzyme essential for the biosynthesis of pyrimidines. In mammalian cell culture, the agent exhibits antiproliferative properties that can be reversed by the addition of uridine. CL 306,293 inhibits the development of the clinical disease in a murine model of immunodeficiency induced by a mixture of LP-BM5 retroviruses. In infected mice, the agent prevents the development of hypergammaglobulinemia, lymphadenopathy, splenomegaly and induction of an IL-2 deficiency. The CD4/CD8 ratio and the number of B cells in the lymph nodes are decreased if the infected animals are treated with CL 306,293. CL 306,293 was more efficacious and potent than 3'-azido-3'-deoxythymidine. The beneficial effects of CL 306,293 observed in this model are most probably related to its antiproliferative properties.

Cytokine gene expression of endothelial cells infected with Trypanosoma cruzi.

Coronary microvascular spasm and platelet hyperreactivity have been implicated in the pathogenesis of Chagas' cardiomyopathy. To clarify further the role of the microvasculature in this disease, alterations in cytokine gene expression due to Trypanosoma cruzi infection of human umbilical vein endothelial cells were examined. Northern blot analysis of total RNA from endothelial cells demonstrated that interleukin (IL)-1 beta, IL-6, and colony-stimulating factor 1 (CSF-1) mRNA expression was absent or minimal in uninfected cells but significantly increased in infected cells. c-sis mRNA levels diminished with increased time of infection. In situ hybridization studies also demonstrated high levels of IL-6 mRNA in individual infected cells. Significant levels of IL-6 and IL-1 beta protein were detected in the supernatants of infected endothelial cells. The serum of an acutely infected individual contained high levels of IL-6 protein, suggesting the potential importance of cytokines secreted by the vascular endothelium in the pathogenesis of Chagas' cardiomyopathy.

Fructose-induced fluorescence generation of reductively methylated glycated bovine serum albumin: evidence for nonenzymatic glycation of Amadori adducts.

In vitro glycation of bovine serum albumin by fructose (fructation) induces fluorescence generation about 10-times faster than glucose (G. Suárez et al. (1989) J. Biol. Chem. 264, 3674-3679). In order to gain further insight into possible mechanisms that would explain this difference, the protein was glycated with either glucose or fructose and then reincubated in the absence of sugars. In contrast to the previous findings, albumin that had been glycated with glucose generated fluorescence at a higher rate during the sugar-free incubation. However, when partially glycated BSA was reincubated with sugars under conditions where de novo glycation was prevented by reductive methylation of amino groups fructose induced fluorescence to a much larger extent than glucose. These results are consistent with the notion of covalent addition of sugars to Amadori groups, the earliest stable products of the Maillard reaction. A chemical pathway is proposed where pyrrolic structures result from the double sugar adducts by aldol condensation and dehydration. These structures might be precursors of fluorophores.

Alterations in the expression of colony-stimulating factor-1 and its receptor during an acute graft-vs-host reaction in mice.

During the course of an acute graft-vs-host reaction in the mouse we observed a progressive increase in the concentration of CSF-1 in serum and liver, peaking at day 14. In contrast, there was a progressive decrease in the splenic CSF-1 concentration. In vivo studies of 125I-CSF-1 uptake and degradation and in vitro studies of 125I-CSF-1 binding by splenic cells demonstrated that within 24 h of the reaction the number of CSF-1 receptor+ cells had increased by 2-fold and their capacity to express the CSF-1 receptor by approximately 3-fold, resulting in a approximately 2.5-fold increase in the splenic clearance of CSF-1 from the circulation. Inasmuch as at 24 h, serum CSF-1 was not significantly altered, these results are suggestive of an increased rate of release of CSF-1 into the circulation early in the response. The splenic CSF-1-receptor bearing cells were in a Mac-1+ fraction that is consistent with a role for CSF-1 in the generation of host-derived splenic macrophages in acute graft-vs-host reaction.

The effect of 3,6-bis(2-piperidinoethoxy) acridine trihydrochloride (CL 246, 738) on acute graft vs host reactions in mice.

3,6-Bis (piperidinoethoxy) acridine trihydrochloride (CL 246, 738) prevented the development of graft vs host (GVH) disease in normal BDF1 mice injected with C57BL/6 parental spleen cells. A single oral dose (50 mg/kg) given on day 0 or day -1 of GVH induction prevented the day 10 GVH-associated suppression of mitogen responsiveness and IL-2 production. The drug was ineffective if given later (days 3-7) in the reaction. The protective effect of CL 246,738 was neutralized by injecting drug-treated GVH mice with antibody to asialo GM-1 (ASGM-1). This suggested that the protective mechanism was not due to a direct effect of the drug on donor cells but rather was achieved indirectly through the activation of host ASGM-1+ cells which then rejected donor lymphocytes. This hypothesis was supported by immunofluorescence which showed that the donor-host chimerism seen in control GVH mice was not found in drug-treated GVH mice. Direct verification of this hypothesis was provided by data which showed that the transfer of CL 246, 738-activated large granular lymphocytes from normal F1 mice can prevent donor-induced immunosuppression in GVH mice. The results suggest that CL 246,738 is a potent immunostimulant which can boost natural resistance of normal unirradiated mice.

Altered plasma membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes.

The intraerythrocytic development of the malaria parasite is accompanied by distinct morphological and biochemical changes in the host cell membrane, yet little is known about development-related alterations in the transbilayer organization of membrane phospholipids in parasitized cells. This question was examined in human red cells infected with Plasmodium falciparum. Normal red cells were infected with strain FCR3 or with clonal derivatives that either produce (K+) or do not produce (K-) knobby protuberances on the infected red cells. Parasitized cells were harvested at various stages of parasite development, and the bilayer orientation of red cell membrane phospholipids was determined chemically using 2,4,6-trinitrobenzene sulphonic acid (TNBS) or enzymatically using bee venom phospholipase A2 (PLA2) and sphingomyelinase C (SMC). We found that parasite development was accompanied by distinct alterations in the red cell membrane transbilayer distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Increases in the exoplasmic membrane leaflet exposure of PE and PS were larger in the late-stage parasitized cells than in the early-stage parasitized cells. Similar results were obtained for PE membrane distribution using either chemical (TNBS) or enzymatic (PLA2 plus SMC) methods, although changes in PS distribution were observed only with TNBS. Uninfected cohort cells derived from mixed populations of infected and uninfected cells exhibited normal patterns of membrane phospholipid organization. The observed alterations in P falciparum-infected red cell membrane phospholipid distribution, which is independent of the presence or absence of knobby protuberances, might be associated with the drastic changes in cell membrane permeability and susceptibility to early hemolysis observed in the late stages of parasite development.

Ligand state of intraerythrocytic circulating HbC crystals in homozygote CC patients.

Whole blood and Stractan-Percoll fractions of blood from splenectomized patients with homozygous hemoglobin C (CC) disease were studied under aerobic and anaerobic conditions. Erythrocytes containing typical CC crystals are found in the densest fraction as documented by freeze-fracture electron microscopy. We report that the intraerythrocytic Hb C circulating crystals are in the oxygenated liganded state as demonstrated by melting upon deoxygenation and by absorption spectroscopy. Furthermore, crystals are more likely to form in cells with low concentration of Hb F. Changes of ligand state (which results in melting of the intraerythrocytic crystal) might be involved in the pathophysiology of this disease, removing the danger of vasoocclusive episodes.

Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes.

We have studied the pathophysiology of the vascular obstruction induced by Plasmodium falciparum-parasitized erythrocytes with the use of an ex vivo microcirculatory preparation perfused with red cells infected with knobless and knobby clones of the FCR-3 strain. We find that parasitized erythrocyte membrane knobs are indispensable for the generation of the circulatory obstruction. Uninfected erythrocytes incubated in culture and erythrocytes infected with early or late forms of the knobless clones or the early forms of the knobby clone all failed to obstruct the microcirculation, although exhibiting various effects on bulk viscosity and peripheral resistance during flow. In contrast, late forms of the knobby clone produced significantly higher peripheral resistance during flow and significant obstruction as detected by changes in time of pressure flow recovery as well as by direct videorecorded microscopic observation. Optical and electron microscopy showed that the adherence of parasitized cells to the endothelium was limited to the venules and involved the knobs in junctions. In addition, we were able to follow the sequence of events during obstruction: initial red-cell adherence to the venular endothelium (sometimes only transitory) followed by progressive recruitment at the venule surface, finally leading to total obstruction that involved parasitized and nonparasitized erythrocytes. Sometimes, retrograde aggregation would extend the obstruction to the capillaries or even precapillary arterioles. These results show that knobs are necessary and sufficient to produce vascular obstruction and that other factors (spleen, immunological, etc.) can only have a modulating role. These results also exclude the possibility that the exclusive adherence to venules is the consequence of "plasma factors" found in the malaric patients.

Plasmodium falciparum: invasion and development in highly parasitized cultures.

We report that synchronized cultures of Plasmodium falciparum with up to 40% parasitized cells can be obtained with the use of low, red blood cell suspensions and only daily replacement of culture medium. These cultures contained not only a reduced proportion of uninfected red cells but also a population of cells with brief and equal time of exposure to culture conditions. Such high parasitemias are desirable for studies of ring-staged parasites (for which enrichment techniques are not available) and late-staged parasites when the manipulations for enrichment are inappropriate or unsuccessful.

The effect of X chromosome inactivation on the inhibition of Plasmodium falciparum malaria growth by glucose-6-phosphate-dehydrogenase-deficient red cells.

Previous data on in vitro culture of Plasmodium falciparum malaria demonstrated that red cell glucose-6-phosphate dehydrogenase deficiency (G6PD-) inhibited parasite growth in deficient hemizygous males. This study investigated the effect of heterozygosity for G6PD- on parasite growth. Blood was obtained from 8 female Sardinian G6PD- heterozygotes with G6PD normal cells ranging from 13% to 60%. For comparison, blood from a G6PD- hemizygous male, containing 100% deficient red cells, was mixed in different proportions with compatible normal blood. In both experiments, parasite growth was inhibited by the presence of deficient cells. In both cases, it was found that the inhibition could be explained by a simple dilution of normal cells by G6PD- cells. Thus, the typical female heterozygote is also protected to a significant extent. When considering the "malaria hypothesis" as it relates to G6PD, protection of the female heterozygote as well as the male hemizygote must be taken into account.

Glucose-6-phosphate dehydrogenase deficiency inhibits in vitro growth of Plasmodium falciparum.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49)-deficient red blood cells from male hemizygotes and female heterozygotes from the island of Sardinia were studied for their ability to support growth in vitro of the malaria-causing organism Plasmodium falciparum. Parasite growth was approximately one-third of normal in both hemi- and heterozygotes for G6PD deficiency. In Sardinians with the beta 0-thalassemia trait, parasite growth was normal except when G6PD deficiency occurred together with the thalassemia trait. The data support the hypothesis that G6PD deficiency may confer a selective advantage in a malarious area; the female heterozygote may be at a particular advantage because resistance to malaria equals that of male hemizygotes, but the risk of fatal hemolysis may be less. However, more female heterozygotes must be studied to confirm this hypothesis. No protective effect of beta 0-thalassemia trait could be demonstrated in vitro.