Cell Cycle Inhibition To Treat Sleeping Sickness.

African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens.IMPORTANCE The development of drugs to treat infections with eukaryotic pathogens is challenging because many key virulence factors have closely related homologues in humans. Drug toxicity greatly limits these development efforts. For pathogens that replicate at a high rate, especially in the blood, an alternative approach is to target the cell cycle directly, much as is done to treat some hematologic malignancies. The results presented here indicate that targeting the cell cycle via inhibition of ribonucleotide reductase is effective at killing trypanosomes and prolonging the survival of infected animals.

Endothelial transmigration by Trypanosoma cruzi.

Chagas heart disease, the leading cause of heart failure in Latin America, results from infection with the parasite Trypanosoma cruzi. Although T. cruzi disseminates intravascularly, how the parasite contends with the endothelial barrier to escape the bloodstream and infect tissues has not been described. Understanding the interaction between T. cruzi and the vascular endothelium, likely a key step in parasite dissemination, could inform future therapies to interrupt disease pathogenesis. We adapted systems useful in the study of leukocyte transmigration to investigate both the occurrence of parasite transmigration and its determinants in vitro. Here we provide the first evidence that T. cruzi can rapidly migrate across endothelial cells by a mechanism that is distinct from productive infection and does not disrupt monolayer integrity or alter permeability. Our results show that this process is facilitated by a known modulator of cellular infection and vascular permeability, bradykinin, and can be augmented by the chemokine CCL2. These represent novel findings in our understanding of parasite dissemination, and may help identify new therapeutic strategies to limit the dissemination of the parasite.

Physiological concentrations of genistein and 17ß-estradiol inhibit MDA-MB-231 breast cancer cell growth by increasing BAX/BCL-2 and reducing pERK1/2.

AIM: The aim of the present study was to identify the mechanism by which genistein and 17ß-estradiol inhibit proliferation of MDA-MB-231 breast cancer cells. MATERIALS AND METHODS: The expression of cell signaling proteins involved in cell apoptosis, proliferation, and survival (BCL-2 associated X protein, BAX; B-cell lymphoma 2, BCL-2; extracellular signal regulated kinase, pERK1/2; and protein kinase B, pAKT) were examined by western blotting, and tested whether these effects correlated with cell proliferation and apoptosis. RESULTS: Compared to the control, 1 µM genistein plus 1 nM 17ß-estradiol significantly increased apoptosis, and the BAX/BCL-2 ratio, with a concomitant decrease in ERK1/2 phosphorylation. High concentrations of genistein (100 µM) both in the presence and absence of 17ß-estradiol also increased apoptosis; however, these changes were not correlated with the BAX/BCL-2 ratio or with phosphorylation of ERK1/2. CONCLUSION: These results suggest that different concentrations of genistein elicit cell responses through different signaling mechanisms. These results are especially relevant in premenopausal women with breast cancer who are on a soy diet.

11q21.1-11q23.3 Is a site of intrinsic genomic instability triggered by irradiation.

The chromosome location, 11q21-23, is linked to loss of heterozygosity (LOH) in multiple tumors including those of breast, lung, and head and neck. To examine the process of LOH induction, the H292 cell line (human muco-epidermoid carcinoma) was irradiated or treated with anti-CD95 antibody, and individual clones isolated through two rounds of cloning. Regions of LOH were determined by screening a suite of eight polymorphic microsatellite markers covering 11p15-11q24 using fluorescent primers and genetic analyzer peak discrimination. LOH induction was observed extending through 11q21.1-11q23.3 in 6/49 of clones surviving 4 Gy and 8/50 after 8 Gy. Analysis of selected clones by Affymetrix 6.0 single nucleotide polymorphism (SNP) arrays confirmed the initial assessment indicating a consistent 27.3-27.7 Mbp deletion in multiple clones. The telomeric border of LOH mapped to a 1 Mbp region of elevated recombination. Whole genome analysis of SNP data indicated that site-restricted LOH also occurred across multiple additional genomic locations. These data indicate that 11q21.1-11q23.3, and potentially other regions of this cell line are sites of intrinsic cell-specific instability leading to LOH after irradiation. Such deletions may subsequently be propagated by genetic selection and clonal expansion.

Genistein in the presence of 17beta-estradiol inhibits proliferation of ERbeta breast cancer cells.

BACKGROUND/AIM: Genistein, a soy component, has been shown to have a biphasic proliferative effect in breast cancer cells, inhibiting in vitro cell proliferation at high concentrations (>10 micromol/l), while stimulating cell proliferation at lower concentrations (<10 micromol/l). However, epidemiological studies have shown an inverse correlation between the intake of genistein and the incidence of breast cancer. One of the possible reasons for this discrepancy could be the differing status of the estrogen receptor (ERalpha and/or ERbeta). Genistein selectively binds to ERbeta with strong affinity and thereby could be a potential chemotherapeutic agent against breast cancer of the ERalpha-negative and ERbeta-positive type. Therefore, the objective of the present study was to determine whether the proliferative effects of genistein were caused by its activity as a selective ERbeta agonist or merely as an antiestrogen. METHOD: This study was carried out in MDA-MB-231 (ERbeta) and T47D (ERalpha and ERbeta) human breast cancer cells. Cell proliferation was determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. The cells were grown in estrogen-starved media and exposed to genistein at different concentrations for 72 h, either in the presence or absence of 17beta-estradiol. RESULTS: A significant decrease in cell proliferation was seen in MDA-MB-231 cells at low concentrations of genistein in the presence of 17beta-estradiol, as compared to genistein alone. In T47D cells, which are known to have a predominance of ERalpha over ERbeta, genistein showed a biphasic cell proliferative response both in the presence and absence of 17beta-estradiol. CONCLUSIONS: Our results suggest that in cells with a predominance of ERalpha, genistein acts as an agonist to ERalpha, and in cells with ERbeta alone, genistein most likely acts as an antiestrogen. Our results also suggest that genistein could be useful as a chemotherapeutic agent in premenopausal women with breast cancer of the ERalpha-negative and ERbeta-positive type.

Rearrangements of the MLL gene are influenced by DNA secondary structure, potentially mediated by topoisomerase II binding.

The location of MLL translocation breakpoints within therapy-related acute myeloid leukemia linked to drugs targeting Topoisomerase II and infant acute leukemia (IAL) are biased toward the intron 11-exon 12 region of MLL, although lacking a comprehensive explanation. To address this, blood samples were taken from breast cancer and lymphoma patients receiving Topoisomerase II inhibitor therapy. Inverse PCR analysis was used to interrogate the exon 12 region of MLL for rearrangements. Eleven of 19 observed translocations showed breakpoint junctions restricted to a single 5 bp location within exon 12. A similarly restricted distribution (11/20 breakpoint junctions) was observed in TK6 cells exposed to either estrogen (linked to IAL) or anti-CD95 antibody. The translocation hotspot was at the 5' edge of a 10-bp tract matched with a perfect palindrome, 101 bp distant. A high stringency Topoisomerase II consensus sequence binding site was noted at the geometric midpoint of the palindromes. Ligation-mediated PCR to screen TK6 cells exposed to anti-CD95 antibody showed 14/37 (38%) of DNA breaks adjacent to the 5' palindrome and 10/37 (27%) at the 3' partner. We propose a model whereby Topoisomerase II facilitates the organization of nuclease-sensitive secondary structures, stabilized by palindrome association, which are prone to rearrangement.

Changes in retinal pigment epithelium related to cigarette smoke: possible relevance to smoking as a risk factor for age-related macular degeneration.

Age-related Macular Degeneration (AMD) is a major cause of central vision loss in the elderly and smoking is a primary risk factor associated with the prevalence and incidence of AMD. To better understand the cellular and molecular bases for the association between smoking and AMD, we determined the effects of Benzo(a)Pyrene (B(a)P), a toxic element in cigarette smoke, on cultured retinal pigment epithelia (RPE) and we examined the RPE/choroid from mice exposed to chronic cigarette smoke. We measured: mitochondrial DNA (mtDNA) damage, phagocytic activity, lysosomal enzymes, exosome markers and selected complement pathway components. In the presence of a non-cytotoxic dose of B(a)P, there was extensive mtDNA damage but no nuclear DNA damage. RPE phagocytic activity was not altered but there were increased lysosomal activity, exocytotic activity and complement pathway components. Retinas from mice exposed to cigarette smoke contained markers for mtDNA damage, exosomes and complement pathway components surrounding Bruch's membrane. Markers for these processes are found in drusen from AMD patients. Thus, smoking may cause damage to mtDNA and increased degradative processes in the RPE. These altered cell biological processes in the RPE may contribute to the formation of drusen in individuals who are cigarette smokers and underlie susceptibility to genetic mutations associated with AMD.

Estrogen treatment induces MLL aberrations in human lymphoblastoid cells.

Epidemiological data indicates increased risk of infant acute leukemia involving MLL gene aberrations with use of oral contraceptives. To determine whether estrogens might be implicated, we examined the effect of estradiol (E2) or 4-OH-E2 in an in vitro model of translocation susceptibility. Genomic DNA from the TK6 human lymphoblastoid cell line was screened by ligation mediated PCR and inverse PCR at a rearrangement hot spot within the MLL breakpoint cluster region to detect DNA aberrations. An increase in DNA double strand breaks was observed within this region after exposure to either E2 or 4-OH-E2. An increase in the frequency of MLL translocations was only found after exposure to E2. Induction of cleavage due to increased activation of apoptotic nucleases was excluded by pre-treatment with the pan-caspase inhibitor, zVAD.fmk. We conclude that concentrations of E2 and 4-OH-E2 that may occur during pregnancy, or during use of oral contraceptives, can cause aberrations of the MLL gene and could thus be a factor in the early events of leukemogenesis occurring in utero.

Autophagy, exosomes and drusen formation in age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of loss of vision in developed countries. AMD is characterized by a progressive degeneration of the macula of the retina, usually bilateral, leading to a severe decrease in central vision. An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate on Bruch's membrane below the retinal pigment epithelium (RPE). Drusen are a risk factor for developing AMD. Some of the protein components of drusen are known, yet we know little about the processes that lead to formation of drusen. We have previously reported increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we used in vitro modeling of increased mtDNA damage. Under conditions of increased mtDNA damage, autophagy markers and exosome markers were upregulated. In addition, we found autophagy markers and exosome markers in the region of Bruch's membrane in the retinas of old mice. Furthermore, we found that drusen in AMD donor eyes contain markers for autophagy and for exosomes. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients.

Autophagy and exosomes in the aged retinal pigment epithelium: possible relevance to drusen formation and age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch's membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch's membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.

Dysfunction of the retinal pigment epithelium with age: increased iron decreases phagocytosis and lysosomal activity.

PURPOSE: Iron accumulation with age in the retinal pigment epithelium (RPE) may be one important source of oxidative stress that contributes to age-related macular degeneration (AMD). Young and old rodent RPE/choroid were compared to assess iron homeostasis during normal aging and the effects of increased iron on the functions of retinal pigment epithelial cells. METHODS: The iron level, mRNA expression, and protein level of iron-regulatory molecules in RPE/choroid were quantitatively compared between young and old animals. To test the effects of increased intracellular iron on the functions of retinal pigment epithelial cells, in vitro ARPE-19 cells were treated with high levels of iron and assessed for phagocytosis activity and lysosomal activity. RESULTS: Iron level was significantly increased in the aged RPE/choroid. Ferritin and ceruloplasmin mRNAs were significantly increased in the aged RPE/choroid, whereas transferrin, transferrin receptor, and ferroportin mRNAs did not change with age. At the protein level, decreased transferrin and transferrin receptor, increased ferritin and ceruloplasmin, and unchanged ferroportin were observed in the aged RPE/choroid. Exposure of ARPE-19 cells to increased iron markedly decreased phagocytosis activity, interrupted cathepsin D processing, and reduced cathepsin D activity in retinal pigment epithelial cells. CONCLUSIONS: The RPE/choroid of aged animals demonstrates iron accumulation and associated alterations in iron homeostasis. Iron accumulation with age may impair the phagocytosis and lysosomal functions of retinal pigment epithelial cells in the aged RPE/choroid. Therefore, age-related changes of iron homeostasis in the RPE could increase the susceptibility of the tissue to genetic mutations associated with AMD.