Expression of Mitochondrial Regulators PGC1α and TFAM as Putative Markers of Subtype and Chemoresistance in Epithelial Ovarian Carcinoma

Epithelial ovarian carcinoma (EOC), the major cause of gynaecological cancer death, is a heterogeneous disease classified into five subtypes. Each subtype has distinct clinical characteristics and is associated with different genetic risk factors and molecular events, but all are treated with surgery and platinum/taxane regimes. Tumour progression and chemoresistance is generally associated with major metabolic alterations, notably altered mitochondrial function(s). Here, we report for the first time that the expression of the mitochondrial regulators PGC1α and TFAM varies between EOC subtypes; furthermore, we have identified a profile in clear-cell carcinoma consisting of undetectability of PGC1α/TFAM, and low ERα/Ki-67. By contrast, high-grade serous carcinomas were characterised by a converse state of PGC1α/TFAM, ERα positivity and a high Ki-67 index. Interestingly, loss of PGC1α/TFAM and ERα was found also in a non-clear cell EOC cell line made highly resistant to platinum in vitro. Similar to clear-cell carcinomas, these resistant cells also showed accumulation of glycogen. Altogether, our data provide mechanistic insights into the chemoresistant nature of ovarian clear-cell carcinomas. Furthermore, these findings corroborate the need to take into account the diversity of EOC and to develop subtype specific treatment strategies.     

Quercetin interaction with the chloroplast ATPase complex

1. Quercetin, a flavonoid which acts as an energy transfer inhibitor in photophosphorylation is shown to inhibit the P-ATP exchange activity of membrane-bound CF1 and the ATPase activity of isolated CF1. Quercetin, affects also the proton uptake in chloroplasts in a manner similar to that of dicyclohexylcarbodiimide. 2. The light-dependent proton uptake in EDTA-treated chloroplasts is stimulated by quercetin. In untreated chloroplasts quercetin has a dual effect: it enhances at pH above 7.5 while at lower pH values it decreases the extent of H+ uptake. Similar effects were obtained with dicyclohexylcarbodiimide. 3. Like quercetin, dicyclohexylcarbodiimide was also found to inhibit the ATPase activity of isolated CF1. 4. Quercetin inhibits uncoupled electron transport induced by either EDTA-treatment of chloroplasts or by addition of uncouplers. Quercetin restores H+ uptake in both types of uncoupled chloroplasts. 5. The mode of action of quercetin and dicyclohexylcarbodiimide in photophosphorylation is discussed, and interaction with both CF1 and F0 is suggested.     

The interaction of N,N'-dicyclohexylcarbodiimide with chloroplast coupling factor 1

1. Incubation of soluble spinach Coupling Factor 1 (CF1) with dicyclohexylcarbodiimide (DCCD) results in the inactivation of the ATPase. The DCCD inactivation is time- and concentration-dependent. Complete inactivation of the CF1-ATPase activity requires the binding of 2 mol of DCCD/mol of CF1. The binding sites of DCCD are located on the beta subunit of CF1. 2. DCCD modification of soluble CF1 eliminates one adenine nucleotide binding site which is exposed by dithiothreitol activation or by incubation with tentoxin. The inactivation of both the ATPase activity and the adenine nucleotide binding site are pH-dependent. The inactivation of both the ATPase activity and the adenine nucleotide binding site are pH-dependent. Half-maximal inhibition occurs at about pH 7.5. 3. The DCCD-modified CF1, reconstituted with EDTA-treated chloroplasts, is fully active is restoring proton uptake but not in restoring ATP synthesis or light-dependent adenine nucleotide exchange.     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1).

Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF1 were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000. In the presence of 1.25 mM MgCl2, 1 mol of ATP or FTP is bound to the latent enzyme, with Kd = 10(-7) or 2 . 10(-7), respectively. The fluorescence emission (lambdamax 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with k1 = 2.4 . 10(4) M-1 . s-1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with K-1 = 3 . 10(-3) S-1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed. The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another. The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 10(6) M-1 . s-1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Defective stress kinase and Bak activation in response to ionizing radiation but not cisplatin in a non-small cell lung carcinoma cell line

We have here examined ionizing radiation (IR)-induced apoptotic signaling in one IR-sensitive small cell lung carcinoma (SCLC) and one resistant non-small cell lung carcinoma (NSCLC) cell line, both harboring mutant p53. In the sensitive SCLC cell line, IR induced conformational modulation of Bak and Bax, mitochondrial depolarization, and nuclear fragmentation. These events were not observed in the IR-resistant NSCLC cell line. However, in the same cells, cisplatin, a DNA-damaging drug, induced Bak and Bax modulation, mitochondrial depolarization, and nuclear fragmentation. Pre-mitochondrial signaling events were examined in order to further characterize the differing IR response. In the SCLC cell line, IR-induced apoptotic signaling was found to involve a MEKK1-related pathway and activation of the stress-activated kinases JNK and p38. In comparison, the NSCLC cell line had higher basal levels of activity of JNK and p38, and IR treatment did not further activate these kinases. However, NSCLC cells were sensitive to Bak modulation and apoptosis induced by a kinase-active mutant of MEKK1. Together, the results delineate a mechanism of IR resistance in NSCLC cells and indicate that IR and cisplatin induce Bak modulation and apoptosis via different pathways.     

Increased apoptosis and increased clonogenic survival of 12V-H-ras transformed rat fibroblasts in response to cisplatin

Mutationally activated Ras is involved in tumor progression and likely also in drug resistance. Using survival, viability and apoptosis assays, we have here compared the cisplatin sensitivities of FR3T3 rat fibroblasts and a 12V-H-ras transformed subline (Ras2:3). Around 24 h after cisplatin treatment Ras2:3 cells showed higher apoptosis levels and lower viability than FR3T3. This increased sensitivity correlated with weaker cisplatin-induced activation of Jun N-terminal kinase (JNK). In contrast to apoptosis assays, colony formation assays showed that Ras2:3 were more resistant to cisplatin than were FR3T3. This was partly due to the increased cisplatin sensitivity of FR3T3 seeded at low densities, as required in colony formation assays. In addition, Ras2:3 cisplatin survivors had a higher relative proliferative capacity. Cell cycle analyses showed that FR3T3 cells initially responded with a dose-dependent G2 arrest, while Ras2:3 accumulated in S-phase. Experiments with an anti-apoptotic mutant of MEKK1 suggested that the apoptotic response of Ras2:3 cells is not specific to the S-phase fraction. In summary, the cisplatin response of ras-transformed fibroblasts is distinct from that of parental cells, in that they show increased apoptosis, a different cell cycle response and increased post-treatment proliferative capacity. The results illustrate the need to carefully consider methods and protocols for in vitro studies on chemotherapy sensitivity.