Selective accumulation of the endoplasmic reticulum-Golgi intermediate compartment induced by the antitumor drug KRN5500

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C(14) unsaturated fatty acid through glycine and to the amino group of adenine. The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferative effect of KRN5500 remains to be elucidated. We have found that acute exposure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug results initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells characterized by numerous intracellular vacuoles. These KRN5500-resistant tumor cells exhibited increased staining with the Golgi stain NBD C(6)-ceramide and the ER-Golgi fluorescent dye BODIPY-brefeldin A, which, unlike the parental drug-sensitive cells, was dispersed throughout the cytoplasm. Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc transferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resistant cell lines while the trans-Golgi marker enzyme, galactosyltransferase, was not. The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-53, a cis-Golgi marker protein of the ER-Golgi intermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtubule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determinant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstrate that KRN5500 alters the distribution of newly synthesized carcinoembryonic antigen within the secretory pathway, including arrest of this N-glycosylated protein in the Golgi of LS-174T colon carcinoma cells.     

gamma-Glutamyl transpeptidase (gamma-GT) and maintenance of thiol pools in tumor cells resistant to alkylating agents

Previous studies from this laboratory have established that acquired resistance of murine L1210 leukemia cells to L-phenylalanine mustard (L-PAM) and other alkylating agents is accompanied by a two-to threefold elevation in their glutathione (GSH) concentration (Biochem. Pharm. 31:121). In an attempt to gain insight into the mechanism by which resistant tumor cells maintain their increased GSH content, we have assessed the possible role of gamma-glutamyl transpeptidase (gamma-GT), a membrane bound enzyme involved in GSH metabolism. These results indicate that the enzyme is present in both sensitive and resistant murine L1210 leukemia cells but that the cellular content of gamma-GT is elevated two-to threefold in L-PAM resistant cells as compared to their sensitive counterparts. This elevation in enzymatic activity correlates well with the increased cellular GSH content in resistant cells. The results of a detailed kinetic analysis of gamma-GT activity indicate that there is no difference, between cell types, in the apparent Km of the enzyme for the gamma-glutamyl donor (L-gamma-glutamyl-p-nitroanilide) or the acceptor (glycylglycine). However, the apparent Vmax is increased two-to threefold in L-PAM resistant tumor cells. Investigation into the role of gamma-GT in the extracellular metabolism of GSH indicates that resistant tumor cells metabolize two-fold more GSH than do sensitive cells and that such metabolism results in a similar difference in the intracellular concentration of cysteine. Results of studies with cellular lysates also indicate a role for the enzyme in the supply of cysteine to the glutathione precursor pool of the tumor cell and in the maintenance of elevated GSH concentrations in cells resistant to alkylating agents.     

Substrate specificity of a high-affinity, monovalent cation-dependent amino acid carrier.

When mammary gland explants from mid-pregnant rats were incubated with insulin (5 μg/ml) and [³H]cortisol (5 μg/ml) for one day, the tissue accumulated 1.69 μg cortisol/g wet tissue. During a second incubation with insulin and prolactin (5 μg/ml), only 20% of the steroid was lost per day. Such retention of glucocorticoid had an important biological consequence: the tissue exposed for one day to insulin and cortisol showed a transient stimulation of casein synthesis during a subsequent, five-day incubation with insulin and prolactin. No casein synthesis was detected, if the first culture medium contained only insulin. In conclusion, mammary gland explants from mid-pregnant rats require a glucocorticoid for casein synthesis, but this requirement may be obscured if the explants are initially incubated in medium containing cortisol, since they are capable of accumulating and retaining this steroid. Similar interpretative difficulties may arise in studies on other steroid-tissue relationships.     

Kinetics of the Anti-oxidant Response to Salinity in the Halophyte Cakile maritima

The effects of NaCl stress on the activity of anti-oxidant enzymes (superoxide dismutase, catalase (CAT),peroxidase (POD),ascorbate peroxidase (APX), monodehydroascorbate reductase, dehydroascorbate reductase (DHAR), and glutathionereductase (GR)), anti-oxidant molecules (ascorbate and glutathione), and parameters of oxidative stress (malondialdehyde(MDA), electrolyte leakage, and H_2O_2 concentrations) were investigated in Cakile maritima, a halophyte frequent along theTunisian seashore. Seedlings were grown in the presence of salt (100, 200, and 400 mmol/L NaCI). Plants were harvestedperiodically over 20 days. Growth was maximal in the presence of 0-100 mmol/L NaCl. At 400 mmol/L NaCl, growthdecreased significantly. The salt tolerance of C. maritima, at moderate salinities, was associated with the lowest values ofthe parameters indicative of oxidative stress, namely the highest activities of POD, CAT, APX, DHAR, and GR and high tissuecontent of ascorbate and glutathione. However, prolonged exposure to high salinity resulted in a decrease in anti-oxidantactivities and high MDA content, electrolyte leakage, and H_2O_2 concentrations. These results suggest that anti-oxidantsystems participate in the tolerance of C. maritima to moderate salinities.     

Selective Accumulation of the Endoplasmic Reticulum–Golgi Intermediate Compartment Induced by the Antitumor Drug KRN5500

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C₁₄ unsaturated fatty acid through glycine and to the amino group of adenine. The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferative effect of KRN5500 remains to be elucidated. We have found that acute exposure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug results initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells characterized by numerous intracellular vacuoles. These KRN5500-resistant tumor cells exhibited increased staining with the Golgi stain NBD C₆–ceramide and the ER–Golgi fluorescent dye BODIPY–brefeldin A, which, unlike the parental drug-sensitive cells, was dispersed throughout the cytoplasm. Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc transferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resistant cell lines while the trans-Golgi marker enzyme, galactosyltransferase, was not. The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-53, a cis-Golgi marker protein of the ER–Golgi intermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtubule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determinant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstrate that KRN5500 alters the distribution of newly synthesized carcinoembryonic antigen within the secretory pathway, including arrest of this N-glycosylated protein in the Golgi of LS-174T colon carcinoma cells.     

Analysis of the cytotoxic effects of light-exposed hepes-containing culture medium

Summary The addition ofN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) to RPMI 1640 medium markedly increases the production of cytotoxic products during exposure of the medium to visible light. The cytotoxicity has been analyzed by measuring uptake of [³H]thymidine by murine thymocytes cultured in preirradiated medium containing 25 mM HEPES. Complete inhibition of thymidine uptake was produced by exposing 50% of the culture medium to light for 3 h before addition of cells. The HEPES-mediated effect requires only that HEPES and riboflavin be exposed to light; other medium constituents are not necessary. Hydrogen peroxide is a principal cytotoxic agent produced in this system. It is demonstrated that most, but not all, of the inhibition of thymidine uptake can be attributed to hydrogen peroxide.     

Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response

Background

Mutations in LRRK2 are related to certain forms of Parkinson’s disease and, possibly, to the pathogenesis of Crohn’s disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses.

Methods

Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA).

Results

The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls.

Conclusions

Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.     

Reconstructing Indian-Australian phylogenetic link

Background  

An early dispersal of biologically and behaviorally modern humans from their African origins to Australia, by at least 45 thousand years via southern Asia has been suggested by studies based on morphology, archaeology and genetics. However, mtDNA lineages sampled so far from south Asia, eastern Asia and Australasia show non-overlapping distributions of haplogroups within pan Eurasian M and N macrohaplogroups. Likewise, support from the archaeology is still ambiguous.     

Pertussis toxin is superior to TLR ligands in enhancing pathogenic autoimmunity, targeted at a neo-self antigen, by triggering robust expansion of Th1 cells and their cytokine production

Microbial products are assumed to play a major role in triggering pathogenic autoimmunity. Recently accumulated data have shown that these products stimulate the immune system by interacting with TLRs, expressed on APCs. To examine the capacity of various TLR ligands to trigger pathogenic autoimmunity, we used a system in which naive CD4 cells, specific against hen egg lysozyme (HEL), are injected into recipient mice expressing HEL in their eyes. Only when stimulated, the naive cells acquire pathogenic capacity and induce ocular inflammation. Seven TLR ligands were tested in this system: lipoteichoic acid/peptidoglycan, zymosan, poly (I:C), LPS, pertussis toxin (PTX), flagellin, and CpG oligodeoxynucleotide. Treatment of recipient mice with HEL alone stimulated proliferation of the transferred cells, but no disease, whereas ocular inflammation did develop in recipient mice coinjected with HEL and any one of the seven TLR ligands. Inflammation induced by PTX surpassed by its severity those induced by all other tested TLR ligands and was accompanied by a dramatic increase in number of the transferred cells that acquired features of effector Th1 lymphocytes. Ocular inflammation and number of transferred cells in recipients injected with PTX and HEL were substantially reduced by treatment with Abs against IFN-gamma or IL-12, thus indicating the role of these cytokines in the PTX effect. Overall, our observations demonstrate that various TLR ligands are capable of triggering pathogenic autoimmunity and that PTX surpasses other microbial products in this activity, by stimulating excessive proliferation and polarization toward Th1 of naive T cells.