Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines.

BACKGROUND: Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. METHODS: Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT-PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. RESULTS: Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. CONCLUSIONS: Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.

Individuality in FGF1 expression significantly influences platinum resistance and progression-free survival in ovarian cancer.

BACKGROUND: Ovarian cancer is frequently advanced at presentation when treatment is rarely curative. Response to first-line platinum-based chemotherapy significantly influences survival, but clinical response is unpredictable and is frequently limited by the development of drug-resistant disease. METHODS: We used qRT-PCR analysis to assess intertumour differences in the expression of fibroblast growth factor 1 (FGF1) and additional candidate genes in human ovarian tumours (n=187), and correlated individuality in gene expression with tumour histology, chemotherapy response and survival. We used MTT assays to assess platinum chemosensitivity in drug-sensitive and drug-resistant ovarian cell lines. RESULTS: Marked intertumour differences in gene expression were observed, with each tumour having a unique gene expression profile. Nine genes, including FGF1 (P=1.7 × 10(-5)) and FGFR2 (P=0.003), were differentially expressed in serous and nonserous tumours. MDM2 (P=0.032) and ERBB2 (P=0.064) expression was increased in platinum-sensitive patients, and FGF1 (adjusted log-rank test P=0.006), FGFR2 (P=0.04) and PDRFRB expression (P=0.037) significantly inversely influenced progression-free survival. Stable FGF1 gene knockdown in platinum-resistant A2780DPP cells re-sensitised cells to both cisplatin and carboplatin. CONCLUSION: We show for the first time that FGF1 is differentially expressed in high-grade serous ovarian tumours, and that individuality in FGF1 expression significantly influences progression-free survival and response to platinum-based chemotherapy.

Blue fluorescent deoxycytidine analogues: convergent synthesis, solid-state and electronic structure, and solvatochromism.

We report the synthesis and photospectroscopic characterisation of intrinsically fluorescent triazole-appended cytidines. Fluorescence was found to be highly dependent on solvent conditions. X-Ray crystallographic data show the proton of the exocyclic amine of the nucleobase and the triazole N(3) engaged in a H-bond.

Human telomerase reverse transcriptase protects hematopoietic progenitor TF-1 cells from death and quiescence induced by cytokine withdrawal.

Telomerase is a complex ribonucleoprotein enzyme that exhibits elevated activity in the majority of cases of human leukemia. We have previously shown that retroviral expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), in human cord blood CD34+ cells leads to an enhanced survival of mature hematopoietic cells. The mechanism for this pro-survival effect is not known. Here, we show that telomerase may play a role in leukemogenesis as a survival factor, independent of its role in maintaining telomere length. Retroviral expression of hTERT in the cytokine-dependent, human hematopoietic progenitor cell line, TF-1, resulted in the survival of cells following the withdrawal of cytokine, with protection from apoptosis, but did not promote unlimited replicative potential. This hTERT-mediated effect on cell survival does not involve Bcl-2 family members, results in accumulation of cells in G1 and appears to operate via autocrine expression of IL-3 and activation of the p53/p21 pathway. Survival in the absence of cytokine stimulation was also observed following retroviral expression of hTERT in normal cord blood CD34+ cells. This study demonstrates a novel pro-survival role for hTERT and may have important implications for the role of hTERT in the pathogenesis of leukemia and drug resistance.

Tamoxifen beyond 5 years--patients' decisions regarding entry to the aTTom trial.

The aim of this study was to assess among a population of women who had taken adjuvant tamoxifen for 5 years, how many were prepared to enter a randomised trial looking at the duration of tamoxifen treatment and what was the preference of those who declined trial entry. There is uncertainty as to the optimum duration of adjuvant tamoxifen and this is the subject of the aTTom (adjuvant Tamoxifen Treatment offer more?) trial in which patients are randomised to continue or stop tamoxifen after 5 years. Patients have been recruited to the aTTom trial in Dundee since 1996 and a record has been kept of all the patients with whom the trial was discussed. Patients who declined trial entry were allowed to choose whether to electively stop or continue tamoxifen. 306 patients were eligible for trial entry of whom 171 (56%) consented to randomisation (82 to continue and 89 to stop). Amongst the 135 (44%) who declined randomisation, 28 (21%) elected to stop tamoxifen treatment, 90 (67%) elected to continue and in 17 (13%) their decision was unclear. These results illustrate that patients eligible for the aTTom trial share our clinical equipoise. A majority (56%) of patients were agreeable to randomisation, but among those who declined, some (67%) preferred to continue, some (21%) to stop tamoxifen. This trial is unusual in that the patients have already experienced the treatment options, so the patients' preferences reflect a truly informed choice.

The role of pro-drug therapy in the treatment of cancer.

The administration of anti-cancer agents is currently associated with significant toxicity and lack of tumour specificity. Prodrugs are being designed to favourably alter the therapeutic index of these agents by improving their efficacy and reducing toxicity. Progress in the development of prodrugs including the cytotoxic agents most commonly used in cancer treatments namely 5-fluorouracil (5-FU), the anthracyclines, paclitaxel and platinum will be described. Many of these agents are at an early stage of development: however, this article will also describe those which have already made an impact in the clinic. It is likely that future improvements in care will come from refinement of the drugs already well established in clinical practice. In addition, this technology could be applied to novel agents with alternative cellular targets such as those involved in angiogenesis or in conferring metastatic potential. Thus, lessons learned with standard drugs may be applicable across a wider spectrum of therapeutics.

Beyond behaviorism: on the automaticity of higher mental processes.

The first 100 years of experimental psychology were dominated by 2 major schools of thought: behaviorism and cognitive science. Here the authors consider the common philosophical commitment to determinism by both schools, and how the radical behaviorists' thesis of the determined nature of higher mental processes is being pursued today in social cognition research on automaticity. In harmony with "dual process" models in contemporary cognitive science, which equate determined processes with those that are automatic and which require no intervening conscious choice or guidance, as opposed to "controlled" processes which do, the social cognition research on the automaticity of higher mental processes provides compelling evidence for the determinism of those processes. This research has revealed that social interaction, evaluation and judgment, and the operation of internal goal structures can all proceed without the intervention of conscious acts of will and guidance of the process.

Biomechanical and biochemical study of a standardized wound healing model.

Standardized protocols were developed for use in a detailed investigation into the biomechanical and biochemical properties of a dermal wound healing model in the rat. The use of a rapid freezing method at -80 degrees C minimized the detrimental effects of freezing on the biomechanical properties of the tissue and also allowed for convenient inter-laboratory collaboration to be performed. The methodology described allowed for the simultaneous and reproducible measurement of tensile strength, collagen cross-linking and proteolytic enzyme activity. Increases in the tensile properties of the tissue with time were consistent with an active process of remodelling process as indicated by changes in the cross-link and enzyme profiles. Initially the granulation tissue was comparatively rich in the keto-imine cross-link hydroxylysino-keto-norleucine, which was later replaced by the aldimine cross-link dehydro-hydroxy-lysinonorleucine. The mature cross-link histidino-hydroxy-lysinonorleucine was not observed within the granulation tissue at any stage and was also absent in aged control skin. A peak of matrix metalloproteinase-9 activity was observed at early timepoints (48 hr) and then decreased rapidly to normal levels and is consistent with an acute inflammatory response. In contrast matrix metalloproteinase-2 activity peaked later (3 days) and then decreased gradually, consistent with its role as one of the predominant enzymes involved in the remodelling process. The results described validate the animal model used and emphasize its potential for use in combined biomechanical and biochemical studies of acute wound healing.

Thalidomide: lack of mutagenic activity across phyla and genetic endpoints.

The human and rabbit teratogen thalidomide has been tested for mutagenicity in a wide range of assays, ranging from bacterial gene mutation assays conducted in vitro to in vivo cytogenetic assays conducted using rabbits, and including a variety of human-derived tissues. Thalidomide was not mutagenic to 6 strains of Salmonella when tested both in the presence and absence of Aroclor-induced rat liver S9 mix. This inactivity was confirmed in strains TA98 and TA100 using a 1-h pre-incubation assay protocol with the same S9 mix (10% S9), and additionally, in strain TA98 using 3 concentrations of S9 (4%, 10% and 30% S9 in S9 mix). Thalidomide was not clastogenic either to cultured human lymphocytes (whole blood cultures, minus S9 mix) or to Chinese hamster ovary (CHO) cells treated in vitro. Further, no cytotoxicity was observed in purified human lymphocytes when exposed to thalidomide up to the limit of its solubility in the medium in the presence and absence of liver S9 from Aroclor-induced pregnant rabbit. The CHO assays were conducted without metabolic activation and in the presence of a variety of sources of auxiliary metabolic activation (PB/beta NP-induced rat liver S9 mix, pooled male and female human liver S9 mix, uninduced and Aroclor-induced pregnant rabbit liver S9 mix and foetal rabbit S9 mix). Thalidomide did not induce micronuclei in isolated human lymphocytes (minus S9 mix) and it was non-mutagenic to mouse lymphoma L5178Y TK+/- cells when tested to the limits of its solubility in the culture medium (+/- S9 mix). No indication of recombinogenic or clastogenic activity was observed for thalidomide when tested in Drosophila. In addition, it failed to induce chromosome aberrations in grasshopper neuroblasts when tested in the presence and absence of Aroclor-induced rat liver S9 mix. Some unusual chromosome morphologies were observed in the grasshopper cytogenetic preparations indicating a potential of thalidomide to interact with chromosomal proteins. However, this potential was not evident in the human lymphocyte micronucleus assay, and thalidomide was apparently not reactive to the proteins of the mouse skin, as it gave negative results in a mouse local lymph node assay for skin sensitizing agents. Thalidomide was inactive in bone marrow micronucleus assays conducted using males and females from two strains of mice, and female New Zealand white rabbits. It is concluded that thalidomide is neither a mutagen nor an aneugen. This conclusion is discussed within the context of the results of earlier mutagenicity studies, the recent claim that thalidomide may be a heritable germ cell mutagen to humans, and the current interest in thalidomide for the treatment of immune system-related diseases.

Mitomycin C effects on cell cycle progression, including inhibition of very late prophase, as seen in living neuroblasts of Chortophaga viridifasciata, with some observations on mitomycin C purity.

Observations were made on living neuroblasts (Nbs) of the grasshopper (Chortophaga viridifasciata) embryo during a 4-h recovery period following 1-h in vitro exposure to 10(-8), 10(-6), and 10(-4) M mitomycin C (MMC). None of these concentrations affected the duration of mid-mitosis (prometaphase, metaphase, anaphase), but one as low as 10(-8) M causes a small reduction in the rate at which Nbs move through the remainder of the cell cycle, primarily by retarding their progress through S. As the concentration is increased there is slower movement through S and also prophase (there are no true G1 and G2 periods in the rapidly dividing Nb: 4-h cell cycle at 38 degrees C). A significant proportion of the cells exposed to 10(-4) M are blocked for 1 or more h at very late prophase, i.e., just before nuclear membrane breakdown. In such retarded prophases the chromosomes resemble c-metaphase chromosomes even though the nuclear membrane remains intact. Mass spectrometry data revealed that one lot of the MMC used contained one or more impurities.

Chromosome fragments and other abnormalities induced by mitomycin C in the neuroblast of Chortophaga viridifasciata.

Mitomycin C (MMC) induces acentric chromosome fragments in the neuroblast (Nb) of the grasshopper embryo (Chortophaga viridifasciata) after acute and chronic exposure to concentrations ranging from 10(-8) to 10(-4) M, the dose response being essentially linear up to 10(-5) M. Because Colcemid is not used in the Nb assay, it was possible to detect two additional effects of MMC: (1) Prolonged retardation of many cells occurs when they reach very late prophase; the chromosomes continue condensing and lose their orderly prophase orientation, and the nuclear envelope becomes increasingly fragile. Such cells, which were observed after both acute and chronic exposure, give the false impression of being c-metaphases when they are fixed and squashed. The frequency of retarded very late prophases and the duration of retardation are related to MMC concentration and time of exposure. A rationale is presented supporting the idea that the events associated with retarded very late prophase result from MMC effects on the nuclear envelope. (2) MMC significantly increases the frequency of Nb's with attenuated centromeres at the beginning of early anaphase, an effect that appears to be caused by a delay in the repulsion of sister chromatids that usually occurs immediately after centromere separation begins.

Preferential decrease in thymus dependent lymphocytes during storage at 4 C in anticoagulant.

Human lymphocytes stored at 4 C either as leukocyte concentrates (LCs) in citrate-phosphate-dextrose (CPD) or as whole blood anticoagulated with CPD show a rapid and marked decrease in the relative and absolute numbers of thymus derived (T) lymphocytes. Determinations were made on cells recoverable on a Ficoll-Hypaque (F-H) gradient. In evacuated LCs, the relative percentage of T cells dropped to less than 10 per cent within 72 hours with a concomitant increase in the relative percentage of bone marrow derived (B) cells to 80 per cent or more. LCs opened to the air and subsequently stored at 4 C displayed an even more precipitous decline in the relative percentage of T cells, reaching a 10 per cent level within 72 hours. The relative percentage of T cells in CPD-anticoagulated whole blood samples stored at 4 C displayed similar decreases, reaching 20 per cent levels within 24 hours. The change in the relative percentage of T cells at the Ficoll-Hypaque interface was shown to reflect a decrease in the total numbers of T cells placed on the F-H gradient with time, since determinations of T and B cell numbers in NH4Cl-treated whole blood showed a 65 to 80 per cent decrease in the numbers of T cells within 24 hours in anticoagulated whole blood held at 4 C. Thus, it may be inferred that the T cell decrease is mediated via some interaction of anticoagulant, storage time, and some component(s) present in both LCs and whole blood.