The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition.

BACKGROUND: Combined targeting of MAPK and PI3K signalling pathways may be necessary for optimal therapeutic activity in cancer. This study evaluated the MEK inhibitors AZD6244 and PD0325901, alone and in combination with the dual mTOR/PI3K inhibitor NVP-BEZ235 or the PI3K inhibitor GDC-0941, in three colorectal cancer cell lines. METHODS: Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively, in HCT116, HT29 and DLD1 cell lines. RESULTS: All MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition; however, GDC-0941 displayed greater synergy in combination with either MEK inhibitor. NVP-BEZ235 exhibited stronger inhibition of 4EBP1 phosphorylation, and similar inhibition of S6 and AKT phosphorylation, compared with GDC-0941. Both PD0325901 and AZD6244 inhibited ERK phosphorylation, and with MEK/PI3K inhibitor combinations inhibition of S6 phosphorylation was increased. The reduced synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared with GDC-0941, may be due to inhibition of mTOR, and the addition of the mTORC1/2 inhibitor KU0063794 compromised the synergy of GDC-0941:PD0325901 combinations. CONCLUSION: These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the combination of specific PI3K inhibitors, rather than dual mTOR/PI3K inhibitors, with MEK inhibitors results in greater synergy.

Identification and evaluation of a potent novel ATR inhibitor, NU6027, in breast and ovarian cancer cell lines.

BACKGROUND: The ataxia telangiectasia mutated and Rad3-related kinase (ATR) has a key role in the signalling of stalled replication forks and DNA damage to cell cycle checkpoints and DNA repair. It has long been recognised as an important target for cancer therapy but inhibitors have proved elusive. As NU6027, originally developed as a CDK2 inhibitor, potentiated cisplatin in a CDK2-independent manner we postulated that it may inhibit ATR. METHODS: Cellular ATR kinase activity was determined by CHK1 phosphorylation in human fibroblasts with inducible dominant-negative ATR-kinase dead expression and human breast cancer MCF7 cells. Cell cycle effects and chemo- and radiopotentiation by NU6027 were determined in MCF7 cells and the role of mismatch repair and p53 was determined in isogenically matched ovarian cancer A2780 cells. RESULTS: NU6027 is a potent inhibitor of cellular ATR activity (IC(50)=6.7 µM) and enhanced hydroxyurea and cisplatin cytotoxicity in an ATR-dependent manner. NU6027 attenuated G2/M arrest following DNA damage, inhibited RAD51 focus formation and increased the cytotoxicity of the major classes of DNA-damaging anticancer cytotoxic therapy but not the antimitotic, paclitaxel. In A2780 cells sensitisation to cisplatin was greatest in cells with functional p53 and mismatch repair (MMR) and sensitisation to temozolomide was greatest in p53 mutant cells with functional MMR. Importantly, NU6027 was synthetically lethal when DNA single-strand break repair is impaired either through poly(ADP-ribose) polymerase (PARP) inhibition or defects in XRCC1. CONCLUSION: NU6027 inhibits ATR, impairing G2/M arrest and homologous recombination thus increasing sensitivity to DNA-damaging agents and PARP inhibitors. It provides proof of concept data for clinical development of ATR inhibitors.

Protein expression of the epsilon subspecies of protein kinase C ceases as Swiss 3T6 fibroblasts increase in cell density even though message for the protein is still present.

We have noted previously that growth of C6 glioma cells from low cell density to confluency and quiescence in serum is accompanied by changes in protein content of different protein kinase C (PKC) subspecies. Here we show that the same occurs as non-contact-inhibiting Swiss 3T6 fibroblasts grow to high density in the presence of serum. Protein expression of PKC subspecies alpha and delta increases as the cells increase in density while that of PKC-zeta remains the same. Unusually, protein expression of PKC-epsilon is completely down-regulated as cells grow beyond about 50% confluency and no PKC-epsilon protein can be detected in 3T6 fibroblasts at high density by Western blotting. However, mRNA for PKC-epsilon is expressed at all stages of fibroblast growth as revealed by RT-PCR. When high-density 3T6 fibroblasts are passaged to low density in fresh medium, re-expression of PKC-epsilon protein is observed within 15 min and becomes down-regulated again as cells become more dense. This very rapid synthesis of PKC-epsilon is not blocked by the transcription inhibitor actinomycin D but is inhibited by cycloheximide. PKC-epsilon has some characteristics of a novel 'early response' protein whose synthesis in newly passaged 3T6 cells is regulated at the translational level.

Ro31-8220 inhibits protein kinase C to block the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells: p70 S6 kinase and MAPKAP kinase-1beta do not function downstream of PKC in activating PLD.

The use of bisindolylmaleimide derivatives of staurosporine as selective inhibitors of protein kinase C (PKC) is in doubt following the report by Alessi [FEBS Lett. 402 (1997) 121-123] that Ro31-8220 and GF109203X are potent in vitro inhibitors of p70 S6 kinase and mitogen-activated protein kinase-activated protein kinase-1beta, as well as of PKC. Here we show that the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells due to phospholipid hydrolysis by phospholipase D (PLD) is not inhibited by rapamycin or PD98059, specific inhibitors respectively of p70 S6 kinase and MAPKK (MEK) and thus of MAPKAP kinase-1beta but is still completely blocked by Ro31-8220. We conclude therefore that p70S6k and MAPKAP kinase-1beta as well as MAPK are not involved in signalling pathways downstream of PKC that regulate phorbol ester-stimulated phospholipid turnover and that the inhibitory action of Ro31-8220 occurs by blocking PKC which regulates at least one pathway to PLD activation. The PI-3 kinase inhibitor, wortmannin, inhibits the phorbol ester-stimulated release of ethanolamine- but not choline-metabolites from C6 cells suggesting that different PLD isoforms regulate the turnover of PtdEth and PtdCho in C6 cells. Both PLD isoforms are activated via PKC but the PtdEth-PLD is also regulated via a wortmannin-sensitive pathway.

Polymorphism of proteins in malaria parasites following mefloquine treatment.

Proteins in malaria parasites (Plasmodium falciparum) isolated from a patient in Thailand before treatment, and after recrudescence of infection subsequent to mefloquine treatment, were compared by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis. Nine 'pre-treatment' and six 'recrudescent' clones were studied. Variants of the enzyme glucose phosphate isomerase were also noted and mefloquine susceptibility of each clone was measured by in vitro tests. The 'pre-treatment' isolate was found to contain at least four genetically distinct clones, all sensitive to mefloquine, while the 'recrudescent' isolate contained at least two other types of clone, both showing increased tolerance to mefloquine. These two more tolerant types of clone differed from all the sensitive ones studied in regard to several different protein variants as shown by 2D-PAGE analysis. It is concluded that at least two (and probably more) genetically distinct clones of parasites with increased tolerance to mefloquine were present in the parasite population before mefloquine treatment was given, and were selected under mefloquine pressure.

Pyrimethamine resistant mutations in Plasmodium falciparum.

Three mutations in Plasmodium falciparum yielding increased resistance to pyrimethamine were obtained following treatment with chemical mutagens and selection in presence of pyrimethamine. From parasite clone TM4/8.2 a mutant, TM4/8.2/4.1, was produced which raised pyrimethamine resistance about 500 times and was found to involve an amino acid change in the DHFR-TS enzyme molecule from Ser108 to Asn108. A clone of another isolate, T9/94, yielded a mutant, T9/94/300.300, raising pyrimethamine resistance about 10 times and involving an amino acid change from Ile164 to Met164. However, another mutant from T9/94, T9/94/M1-1(b3), although it raised the pyrimethamine resistance 100 times, did not involve any changes in the coding sequence of the DHFR-TS gene, but resulted in the production of about twice as much DHFR-TS enzyme as the original clone T9/94. No amplification of the DHFR-TS gene was detected. It is concluded that changes in pyrimethamine resistance of malaria parasites may arise in at least 2 ways: (1) by structural changes in the DHFR domain of the DHFR-TS gene (as previously found by other workers); (2) by other changes, possibly affecting the expression of the DHFR-TS gene. The relative importance of these 2 mechanisms in causing resistance in wild populations of P. falciparum is discussed.

Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests.

In vitro drug resistance tests of ten isolates of Plasmodium falciparum from three different collection points in Central Thailand have been carried out, and the results compared with those of similar tests with a drug-sensitive West African isolate. Judged by concentration of drug tolerated, the Thai isolates appeared to be about 10 times as resistant to chloroquine, and usually about 10(5) times as resistant to pyrimethamine, as the African isolate. A little variation amongst the Thai isolates was detected.

Variability in drug susceptibility amongst clones and isolates of Plasmodium falciparum.

Heterogeneity within isolates of Plasmodium falciparum in regard to drug susceptibility is described from studies with three Thai isolates and some clones derived from them. One isolate (T9), which before cloning was resistant in vitro to chloroquine and pyrimethamine, contained a diverse assortment of clones, some of which were sensitive to one or other, or both, of these drugs. Another isolate (CH150) was initially sensitive to mefloquine in vitro, but, on recrudescence of infection in the patient, developed a number of clones all of which had diminished susceptibility to mefloquine. Drug pressure in a laboratory culture of CH150 resulted in a similar change in susceptibility. Hence resistant clones are thought to have been present as a minority component of the original isolate CH150. On testing uncloned isolates at different times after growth in culture, drug susceptibility showed considerable variability, but clones remained stable. Reaction in vitro of these isolates to some other drugs (amodiaquine, Fansidar, quinine) is also described, and the results are discussed in relation to changes in drug resistance of malaria parasites which may occur in laboratory cultures and under field conditions.

Susceptibility of Plasmodium falciparum to five drugs: an in vitro study of isolates mainly from Thailand.

This paper describes the results of testing the susceptibility of 60 isolates of the human malaria parasite Plasmodium falciparum from Thailand, and single isolates from five other countries, to five drugs: chloroquine, pyrimethamine, quinine, mefloquine and amodiaquine. The Thai isolates were obtained from patients in three different regions of the country (Chantaburi, Songkhla and Mae Sod), and were first grown in culture by the Trager-Jensen candle-jar technique. Samples were then exposed to a range of concentrations of the five drugs, in Falcon microtest culture wells, for 72 hours, with daily changes of medium (with or without added drug solutions). Presence or absence of parasites was then determined by microscope observations on thin-film Giemsa-stained preparations. Most Thai isolates showed a minimum inhibitory concentration (MIC) for chloroquine of 10(-6) M or higher, and were classified as highly resistant, though one cloned isolate was as sensitive to this drug as a chloroquine-sensitive isolate from West Africa. Similarly most Thai isolates showed a very high resistance to pyrimethamine (MIC 10(-4) M to 10(-6) M), but a few clones were sensitive (MIC 10(-9)) to it. Susceptibility to quinine showed some variation (MIC varied between 10(-6) M and 10(-8) M), and some isolates were thought to be incapable of responding to a therapeutically permissible dose of this drug. Little variation was found in the reaction of any of the isolates to mefloquine or amodiaquine, and by the in vitro technique used in this study, it was found that chloroquine-resistant and chloroquine-sensitive isolates were equally susceptible to amodiaquine. In general the survey showed the existence of a marked correlation between development of drug resistance of Plasmodium falciparum and the extent to which a given drug had been used in Thailand.

Enzyme typing of some isolates of Plasmodium falciparum from Thailand.

One hundred and eighty nine isolates of Plasmodium falciparum collected in Thailand, and eleven originating from Cambodia, have been typed by starch-gel electrophoresis of six enzymes (GPI, LDH, GDH, PGD, ADA, PEPE). Substantial polymorphism was found only with GPI. Occasional variants occurred with ADA, while the other four enzymes appeared to be invariant by the tests used. The results are compared with those of similar studies on African isolates, and lead to the provisional conclusion that P. falciparum isolates from different different endemic areas constitute a single, world wide species, containing potentially interbreeding individual organisms.

Evidence of increased chloroquine sensitivity in Thai isolates of Plasmodium falciparum.

Sensitivity of Thai isolates of Plasmodium falciparum to chloroquine collected over the years 1978-1986 was measured by two methods: (i) by growth of previously cultured isolates for 72 h in presence of drug, and (ii) by the WHO standard in vitro microtest. During this period there were signs of a gradual increase in drug sensitivity, coinciding with the withdrawal of chloroquine for treatment of falciparum malaria in Thailand.

Electrophoretic variants of enzymes in isolates of Plasmodium falciparum, P. malariae and P. vivax from Thailand.

A new electrophoretic variant of glucose phosphate isomerase (GPI), which we now denote GPI-3, has been found in isolates of Plasmodium falciparum from 6 patients, all of whom acquired the infection in the same region (in or near Prachinburi province) of Thailand. In other regions, from which 453 isolates have been tested, only GPI-1 and/or GPI-2 have been found. Two isolates of P. malariae from patients at Kanchanaburi showed a band of GPI activity on cellulose acetate gels at a cathodal position quite distinct from that of any previously known GPI variants in other human malaria parasites. Thirty-nine isolates of P. vivax from 3 regions of Thailand have been examined for variants of GPI and lactate dehydrogenase (LDH). Three forms of GPI were found, corresponding approximately in band positions to GPI-1, 2 and 3 of P. falciparum. The position of the band of LDH activity in P. vivax was the same in all the isolates examined, and different from that of LDH-1 in P. falciparum.

Clonal diversity in a single isolate of the malaria parasite Plasmodium falciparum.

Clones of an isolate of Plasmodium falciparum from Mae Sod (Thailand) were prepared by a dilution procedure. Some of the parasite cultures thus obtained have been typed for the following characters: (i) electrophoretic variants of three enzymes; (ii) susceptibility to chloroquine and pyrimethamine; (iii) antigen diversities recognized by ten strain-specific monoclonal antibodies; (iv) presence or absence of knobs on infected erythrocytes and (v) two-dimensional PAGE variants of seven proteins. Amongst the clones there was variation involving each of these five characters. At least seven different types of clones were found in ten cultures produced by dilution. The amount of phenotypic variation within a single isolate has thus been shown to be surprisingly great. Variations in drug susceptibility and antigens are considered to be particularly important in view of their relevance to anti-malarial treatments.

Fever hospitals in counties Armagh and Down: 1817-39.

This paper outlines the provision for fever patients, (other than those suffering from cholera during the epidemic of 1832-34), in counties Armagh and Down in the two decades prior to the introduction of the Poor Law to Ireland. Possible causes of fever and the numbers of patients treated are discussed. The establishment and location of fever hospitals and the state of the premises are considered and an assessment of the contribution of these institutions to the development of medical provision in the early nineteenth century is also provided.

Dispensaries in counties Armagh and Down in the pre-Famine years.

This paper traces the development of dispensaries in the counties of Armagh and Down in the decades prior to the Great Famine. It examines the number and distribution of dispensaries and discusses their management, finance and daily administration. The role of dispensary doctors, their conditions of employment and the diseases which they treated are also considered.

Treating Ulster's rural poor: the county infirmaries of Armagh and Down 1766-1851.

This paper considers the role of county infirmaries in providing health care for the inhabitants of two counties in south-east Ulster. It traces the establishment and management of these institutions from their beginnings shortly after the passing of the Infirmaries Act (1765) to the middle of the nineteenth century. From the available evidence, the accommodation, staff, patient numbers and diet of the infirmaries are considered and an assessment of their efficacy in offering a valuable service to their communities is discussed.