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.

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.

Plasmodium falciparum: gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine.

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22-46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed.

Signalling pathways regulating the dephosphorylation of Ser729 in the hydrophobic domain of protein kinase Cepsilon upon cell passage.

We have recently demonstrated that in quiescent fibroblasts protein kinase C (PKC) epsilon(95) is phosphorylated at Ser(729), Ser(703), and Thr(566) and that upon passage of quiescent cells phosphorylation at Ser(729) is lost, giving rise to PKCepsilon(87). Ser(729) may be rephosphorylated later, suggesting cycling between PKCepsilon(87) and PKCepsilon(95). Here we show that the dephosphorylation at Ser(729) is insensitive to okadaic acid, calyculin, ascomycin C, and cyclosporin A, suggesting that dephosphorylation at this site is not mediated through protein phosphatases 1, 2A or 2B. We demonstrate that this dephosphorylation at Ser(729) requires serum and cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine, and Ro-31-8220. These results suggest that the phosphorylation status of Ser(729) in the hydrophobic domain at Ser(729) is regulated independently of the phosphorylation status of other sites in PKCepsilon, by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser(729).

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.

Ruptured intrahepatic aneurysm as the initial manifestation of polyarteritis nodosa.

polyarteritis nodosa (PAN) is characterized by panarteritis involving all layers of the vessel wall of medium and small arteries and adjacent veins leading to thrombosis and aneurysmal dilatation. We present the case of a 21-year-old Caucasian man with an acute abdomen with a massive gastrointestinal (GI) hemorrhage caused by a large intrahepatic aneurysmal rupture needing surgery. This was the initial manifestation of underlying PAN, which had not been diagnosed before the event. This patient's postoperative course was complicated by continued hemorrhage prompting further diagnostic abdominal arteriography, which revealed multiple art-eurysms along the superior mesenteric artery. The aneurysmal wall biopsy revealed lymphocytic infiltrates, scattered giant cells, and minimal necrosis. A diagnosis of PAN was entertained based on arteriographic findings, although no pathognomonic demonstration of multiple visceral aneu-rysms is a characteristic finding of PAN, The patient survived the catastrophic event, and treatment with corticosteroids was initiated, which led to an uncomplicated hospital stay. Furthermore, outpatient follow ups showed continued clinical improvement with corticosteroid therapy and a repeat angiogram performed elsewhere demonstrated resolution of most of the aneurysms. Our case represents an unusual initial clinical manifestation of PAN and is one of the few cases reported with a torrential (GI) hemorrhage resulting from a rupture of an intrahepatic aneurysm, which is associated with increased mortality. The clinical significance of abdominal pain is not always apparent. It may occur in the absence of any gross intra-abdominal lesions, or, as in our patient, may herald an acute abdominal catastrophe. The clinician should consider PAN in the differential diagnosis of a massive GI hemorrhage. It is also important to note that early diagnosis followed by aggressive treatment can be lifesaving.

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.

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.

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.

Metastatic choriocarcinoma: correlation of MRI, CT, and angiography.

We have described a 46-year-old woman with metastatic choriocarcinoma, anemia, and a liver mass. She subsequently had widespread metastatic disease and died despite chemotherapy. Without an antecedent history of pregnancy, her clinical presentation posed a diagnostic dilemma. This case emphasizes the importance of including choriocarcinoma in the differential diagnosis of liver masses in women of childbearing age and of correlating CT, MRI, and angiographic findings.

Pneumothorax and bronchopleural fistula during treatment of allergic bronchopulmonary aspergillosis.

A man with allergic bronchopulmonary aspergillosis had pneumothorax and bronchopleural fistula while impacted mucus was being successfully dislodged from bronchiectatic airways. We believe this is the first report of pneumothorax with bronchopleural fistula in a patient with active ABPA, and we have proposed a mechanism to explain the development of this complication.

Aluminium intoxication in undialysed adults with chronic renal failure.

The dialysis encephalopathy syndrome (DES) consists of altered mental status, communication difficulty, seizures and myoclonus. It has been attributed to elevated serum aluminium (A1) levels. Two undialysed patients with chronic renal failure who presented with the characteristic syndrome are reported. The first, a 48 year old female, had used A1 containing phosphate binders for two years. Her serum A1 level was 25.34 mumol/L. Despite treatment with desferoximine and dialysis, she died. Necropsy revealed elevated A1 levels in the cerebral cortex (19 mcg/gm) and spongioform change in the outer three cortical layers. The second patient, a 46 year old woman, had a serum A1 of 8.70 mumol/L. She had never taken A1 containing phosphate binders but had taken several grams/day of citrate for at least six months. Treatment with haemodialysis and discontinuation of the citrate produced a resolution of symptoms and return of the A1 level to normal. During two years of haemodialysis there has been no recurrence.

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.