The MEK1/2 inhibitor, selumetinib (AZD6244; ARRY-142886), enhances anti-tumour efficacy when combined with conventional chemotherapeutic agents in human tumour xenograft models.

BACKGROUND: The Ras/RAF/MEK/ERK pathway is frequently deregulated in cancer and a number of inhibitors that target this pathway are currently in clinical development. It is likely that clinical testing of these agents will be in combination with standard therapies to harness the apoptotic potential of both the agents. To support this strategy, it has been widely observed that a number of chemotherapeutics stimulate the activation of several intracellular signalling cascades including Ras/RAF/MEK/ERK. The MEK1/2 inhibitor selumetinib has been shown to have anti-tumour activity and induce apoptotic cell death as a monotherapy. METHODS: The aim of this study was to identify agents, which would be likely to offer clinical benefit when combined with selumetinib. Here, we used human tumour xenograft models and assessed the effects combining standard chemotherapeutic agents with selumetinib on tumour growth. In addition, we analysed tumour tissue to determine the mechanistic effects of these combinations. RESULTS: Combining selumetinib with the DNA-alkylating agent, temozolomide (TMZ), resulted in enhanced tumour growth inhibition compared with monotherapies. Biomarker studies highlighted an increase in γH2A.X suggesting that selumetinib is able to enhance the DNA damage induced by TMZ alone. In several models we observed that continuous exposure to selumetinib in combination with docetaxel results in tumour regression. Scheduling of docetaxel before selumetinib was more beneficial than when selumetinib was dosed before docetaxel and demonstrated a pro-apoptotic phenotype. Similar results were seen when selumetinib was combined with the Aurora B inhibitor barasertib. CONCLUSION: The data presented suggests that MEK inhibition in combination with several standard chemotherapeutics or an Aurora B kinase inhibitor is a promising clinical strategy.

[Involvement of the IGF system in the pathogenesis of adrenocortical tumors]. / Anomalies du système des IGFs dans les tumeurs cortico-surrénaliennes.

Adrenocortical carcinoma remains a challenge for the therapeutist; prognosis is ominous. Various abnormalities playing a pathogenetic role have been recently described in adrenocortical tumors. Among them, dysregulation of the IGF system and imprinting mistakes at the 11p15 locus play a determining role in malignant transformation of adrenocortical cells. These markers of the malignant phenotype might markedly improve our diagnosis and prognosis abilities.

Evaluation of plasma insulin-like growth factor binding protein-2 as a marker for adrenocortical tumors.

OBJECTIVE: Recent studies have pointed to the role of the IGF system in the pathogenesis of adrenocortical tumors, and it was shown recently that malignant adrenocortical tumors exhibit a high insulin-like growth factor binding protein (IGFBP)-2 content. Circulating markers specific for adrenocortical carcinoma are needed and the aim of this study was to evaluate plasma IGFBP-2 as a marker for these malignant tumors. METHODS: Plasma IGFBP-2 was determined in 51 patients referred to our institutions for adrenocortical tumors. Fifteen patients were in complete remission (group 1), eight patients had preoperative localized tumors (group 2) and 28 patients had metastatic tumors (group 3). Thirty-six healthy volunteers constituted a control group. RESULTS: There was no significant difference in plasma IGFBP-2 concentration between healthy controls and patients with complete remission or localized tumors. In contrast, patients with metastatic disease had significantly higher IGFBP-2 plasma levels than the control group (P<0.001). IGFBP-2 levels in patients with metastatic disease were inversely correlated with survival (R2=0.308; P=0.0026). In patients with localized tumors, there was no correlation between plasma IGFBP-2 concentration and tumor size or histological features. Analysis of individual IGFBP-2 concentrations showed that five patients (17.8%) with metastatic tumors had normal IGFBP-2 levels and two patients (13.3%) in complete remission had high plasma IGFBP-2 levels. The influence of nutrition, hormone secretion and treatment on IGFBP-2 levels was examined. Nutritional status was evaluated by determining IGF-I levels and was found to be normal in 16 patients (61.5%) with high IGFBP-2 levels, suggesting that malnutrition was not responsible for the high IGFBP-2 concentrations in these patients. IGFBP-2 levels did not differ significantly according to tumor secretion or mitotane treatment. In a follow-up study, plasma IGFBP-2 concentration remained stable in patients with complete remission or stabilized disease and was a late marker of tumor progression in patients with progressive metastatic disease. CONCLUSIONS: These results indicate that plasma IGFBP-2 is elevated in patients with malignant adrenocortical tumors and that the major factor affecting IGFBP-2 levels in these patients is tumor stage. However, plasma IGFBP-2 was less sensitive than expected for a tumor marker, which may limit its value in the diagnosis and follow-up of adrenocortical carcinoma.

Establishment and characterization of a human adrenocortical carcinoma xenograft model.

Adrenocortical carcinomas are rare malignant tumors. They have a poor prognosis, as they are often diagnosed late and are usually resistant to chemotherapy. The lack of a suitable animal model for these tumors has been a major obstacle to the evaluation of new therapeutic agents. The aim of this study was to establish and characterize xenografts of the human adrenocortical carcinoma NCI H295R cell line as a model of adrenocortical carcinoma for future therapeutic trials. This cell line was sc injected (6 x 10(6) cells) into nude mice (n = 20). Solid tumors were locally measurable after 45 days at 90% of the inoculation sites. The xenografts were similar histologically to the original adrenocortical carcinoma from which the cell line was derived. The xenografts precisely reproduced the dysregulation of the insulin-like growth factor (IGF) system [overexpression of the IGF-II and IGF-binding protein-2 (IGFBP-2) genes] typical of adrenocortical carcinoma. Similarly to adrenocortical carcinomas, human IGFBP-2 (but not IGF-II) was secreted in mouse plasma. We analyzed steroid production (cortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, delta4-androstenedione, 11-deoxycortisol, corticosterone, and testosterone). Xenografts produced all three class of steroids, with the preferential production of androgens of the delta4 pathway. The H295R xenograft model is a good model of human adrenocortical carcinoma, as it mimics dysregulation of the IGF system usually found in these tumors. It also produces IGFBP-2 and steroids that can be used as tumor markers. This model may therefore be useful for evaluating therapeutic agents.

Alterations of the 11p15 imprinted region and the IGFs system in a case of recurrent non-islet-cell tumour hypoglycaemia (NICTH).

BACKGROUND AND OBJECTIVE: Non-islet-cell tumour hypoglycaemia (NICTH) is a rare disorder and has been explained by oversecretion of non mature IGF-II and dysregulation of the IGFs sytem. The mechanisms responsible for tumoural IGF-II overexpression in NICTH have been rarely studied. We report an extensive study of IGF-II and IGFBPs as well as chromosome 11p15 gene expression regulation in a case of a pleural fibrosarcoma in a 63-year-old patient presenting with NICTH. METHODS AND RESULTS: Abnormal high molecular weight precusor forms of IGF-II were present in the patient's serum and were associated with dramatic alterations in the circulating levels of IGF-I, IGF-II and their binding proteins, as well as an inhibition of the somatotroph axis. These alterations returned to normal following complete surgical removal of the tumour. No structural change in chromosome 11p15 region was apparent in the tumour. However, dysregulation of this imprinted region was demonstrated, leading to the loss of imprinting of the IGF-II gene associated with high IGF-II expression, and by contrast decreased level of expression of H19 and p57KIP2 genes. Recurrence of the tumour four years latter was not associated with hypoglycaemia or changes in the levels of circulating IGFs or IGFBPs, despite IGF-II overexpression by the tumour. This suggests that a large tumour volume is required to reach high enough levels to cause changes in the levels of circulating IGFs and IGFBPs, and to cause hypoglycaemia. CONCLUSION: These results suggest that a dysregulation of gene expression and imprinting of chromosome 11p15 region is associated with tumour growth and IGF-II overexpression in non-islet-cell tumour hypoglycaemia.

Fibroblast growth factor-2 inhibits the maturation of pro-insulin-like growth factor-II (Pro-IGF-II) and the expression of insulin-like growth factor binding protein-2 (IGFBP-2) in the human adrenocortical tumor cell line NCI-H295R.

The IGF system is thought to play a major role in adrenocortical tumorigenesis. In this study, we used the NCI H295R cell line as a model to investigate the effects of fibroblast growth factor-2 (FGF-2), a potent mitogen for normal adrenal cells, on the proliferation and on the expression of the IGF system in cultured adrenocortical tumor cells. Three immunoreactive FGF-2 isoforms of molecular masses 18, 22, and 24 kDa were detected in H295R cell extracts. Recombinant human FGF-2 stimulated the proliferation of adrenocortical tumor cells in a dose- and time-dependent manner, with a maximal effect at a concentration of about 1 ng/ml. Treatment of H295R cells with 10 ng/ml FGF-2 for 7 days had no significant effect on IGF-II messenger RNA levels. However, a marked increase in levels of intracellular IGF-II protein was detected by immunoblotting. In contrast, FGF-2 induced a marked decrease in the amount of IGF-II protein secreted, with the disappearance of mature IGF-II and secretion of higher molecular forms of the growth factor, suggesting modifications of IGF-II processing. Cell cultures in the presence of brefeldin A (1 microg/ml), a specific inhibitor of protein secretion, suggested that FGF-2 did not increase IGF-II synthesis but instead inhibited the secretion of pro-IGF-II from H295R cells, thereby impairing the final steps of IGF-II processing to the mature 7.5-kDa peptide. At the same concentrations, FGF-2 also decreased both IGFBP-2 messenger RNA and secreted protein, which might increase IGF-II bioavailability. No proteolysis of IGFBP-2 was detected in FGF-2-conditioned medium. Altogether, these results indicate that FGF-2 is mitogenic for NCI H295R tumor cells and regulates the expression of both IGF-II and IGFBP-2 in this tumor model. Moreover, this study shows a novel effect of FGF-2, by which this growth factor modulates the processing of pro-IGF-II.

High expression of cyclin E and G1 CDK and loss of function of p57KIP2 are involved in proliferation of malignant sporadic adrenocortical tumors.

Maternal loss of heterozygosity (LOH) of the 11p15 region and overexpression of the insulin-like growth factor (IGF)-II gene are associated with the malignant phenotype in sporadic adrenocortical tumors. In the imprinted 11p15 region, the p57KIP2 gene is maternally expressed and encodes a cyclin-dependent kinase (CDK) inhibitor involved in G1/S phase of the cell cycle. We hypothesized that maternal LOH in malignant adrenocortical tumors could be responsible for loss of p57KIP2 gene expression and, thus, could favor progression through the cell cycle. We investigated 3 normal adrenals, 31 adrenocortical tumors [11 tumors with normal expression of the IGF-II gene (mainly benign) and 20 with IGF-II gene overexpression (mainly malignant)], and the human adrenocortical tumor cell line NCI H295R for expression of the p57KIP2 gene, G1 cyclins (cyclin D2 and E) and G1 CDK (CDK2, CDK3 and CDK4) protein contents and for kinase activity of G1 cyclin-CDK complexes. The expression of p57KIP2, G1 cyclins, and G1 CDKs in benign tumors was similar to that in normal adrenal tissues, as were kinase activities of G1 cyclin-CDK complexes. By contrast, abrogation of the p57KIP2 gene expression and increased expression of G1 cyclins (cyclin E) and G1 CDKs (CDK2 and CDK4) were associated with high activity of G1 cyclin-CDK complexes in malignant tumors and in the H295R cell line. These data suggest that the p57KIP2 gene might act as a tumor suppressor gene in adrenocortical tumors. Maternal LOH with duplication of the paternal allele or pathological functional imprinting of the 11p15 region are responsible for loss of expression of the p57KIP2 gene and increased expression of the IGF-II gene. Consequently, both events favor cell proliferation in malignant adrenocortical tumors.

African AIDS conference offers hope for the future.

PIP: AIDS in Africa is killing more people than all of Africa's armed conflicts during this century--this is what delegates heard at the 11th International Conference on AIDS and STDs in Lusaka, Zambia, September 12-16, 1999. The epidemic has been contained in the rich Northern Hemisphere. But why the difference? Delegates considered many reasons, such as war, crumbling health infrastructure, increasing unemployment, and poverty. The conference concentrated on the impact of HIV/AIDS on communities, and on socioeconomic, ethical, political, and legal factors. Only one of the five sessions was devoted to basic science and clinical care. There was realism concerning the limited role of antiretroviral therapy and excitement concerning improved prevention of parent-to-child transmission, especially if single-dose nevirapine proves effective. Delegates emphasized the need for more research and development of vaccines, the female condom, and vaginal virucides, all of which are commercially unattractive to drug companies. Callisto Medavo, World Bank vice-president, launched ACT-Africa, which promises extra resources, enhanced treatment, and more technical support. Together these should assist African leaders and mobilize civil society and the private sector to intensify action against HIV/AIDS. Peter Piot, executive director of UNAIDS, also emphasized the vital importance of sustained political involvement at the highest level to contain the pandemic and prevent this regional crisis from becoming a global catastrophe.^ieng

Autocrine role of IGF-II in proliferation of human adrenocortical carcinoma NCI H295R cell line.

In adrenocortical tumors, the malignant phenotype is associated with rearrangements (paternal isodisomy) at the 11p15 locus and IGF-II gene overexpression, strongly suggesting that the IGF system is a major determinant of adrenocortical tumor progression. The aim of this study was to validate an in vitro model for investigating the involvement of the IGF system in adrenocortical tumorigenesis. We analyzed the production of IGF mRNA and proteins, IGF-binding proteins (IGFBPs) and IGF receptors by the NCI H295R cell line, which is derived from a human adult adrenocortical carcinoma. H295R cells were shown to proliferate for a long period (26 days) in the absence of serum or any added growth factor. Northern blot analyses showed high IGF-II mRNA contents in H295R cells. The cells secreted large amounts of IGF-II protein (14 ng/10(6) cells per 48 h) although no IGF-I protein was detected. Western ligand blot analyses of conditioned media detected the presence of large amounts of a 34 kDa protein, which was identified as IGFBP-2 by immunoblotting. The presence of high-affinity binding sites for IGF-I and IGF-II on H295R cells was shown by binding experiments using radiolabeled IGFs and confirmed by reverse transcription PCR analyses showing type 1 and type 2 IGF receptors. Proliferation of H295R cells was inhibited by anti-IGF-II antibody (45%) and by anti-type 1 IGF receptor antibody (53%) indicating that IGF-II is an autocrine growth factor for these cells and that its effects are, at least in part, mediated by the type 1 IGF receptor. These findings confirm the involvement of the IGF system in adrenocortical tumors and suggest that the H295R cell line is a suitable in vitro model for studying the molecular mechanisms of adrenocortical tumor proliferation.

Increased levels of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 are associated with malignancy in sporadic adrenocortical tumors.

In adrenocortical tumors, malignancy is strongly associated with insulin-like growth factor II (IGF-II) gene overexpression and abnormalities at the 11p15 locus, suggesting a role for this growth factor in adrenocortical tumorigenesis. To further investigate this role, the IGF/IGF-binding protein (IGFBP) system was analyzed in 18 adrenocortical tumors, classified into 2 groups on the basis of their IGF-II messenger ribonucleic acid (mRNA) content (group 1, normal IGF-II mRNA content, mostly benign tumors; group 2, high IGF-II mRNA content, mostly malignant tumors). Group 2 tumors contained 10 times more IGF-II protein than group 1 tumors or normal adrenal tissue (P < 0.001), indicating efficient translation of IGF-II mRNA in malignant tumors. Western ligand blotting detected various functional IGFBPs in normal adrenocortical glands and tumors: a doublet of 39-42 kDa identified by immunoblotting as IGFBP-3, a band at 32 kDa, and bands at 29-30 and 24 kDa. Total IGFBP-3 protein levels were similar in the two groups of tumors. By contrast, malignant tumors differed from benign ones by specific expression of the 32-kDa IGFBP. Immunoblotting identified this 32-kDa band together with a proteolytic fragment of 25 kDa as IGFBP-2, and quantitative analysis showed significantly higher levels of total IGFBP-2 in malignant tumors than in benign tumors (P < 0.001). Despite enhanced levels of IGBP-2 protein in malignant tumors, no increase in IGFBP-2 mRNA levels was detected, suggesting post-transcriptional regulation of this IGFBP. These results confirm the major role of IGF-II in adrenocortical tumorigenesis and suggest that IGFBP-2 may be a regulator of IGF-II proliferative effects in this tumor system.

A flexible loading dose schedule for warfarin therapy.

A flexible loading dose schedule for inducing anticoagulation with warfarin was assessed in 31 consecutive patients. 55% reached the therapeutic range (prothrombin ratio between 2 and 4:1) by Day 2 (40 hours after the first dose) and this figure rose to 77% on Day 3 and to 87% on Day 4. All patients had a PTR between 1.7 and 4.2 on Day 5. Patients with evidence of cardiac failure and abnormal liver function, and those taking medications known to interact with warfarin required lower doses and ran a higher PTR when compared with the total group of patients. This schedule offers a useful means of safely and rapidly inducing warfarin therapy in all patients.

The nature of iron released by resident and stimulated mouse peritoneal macrophages.

Mouse peritoneal macrophages were allowed to ingest 59Fe, 125I-labelled transferrin-antitransferrin immune complexes, and the release of 59Fe and degraded transferrin was studied. Some iron was released as ferritin, but a major portion was bound by bovine transferrin present in the culture medium, which contained fetal calf serum. If the medium was saturated with iron prior to incubation with the cells, little of the released iron was then bound by transferrin but appeared either as a high molecular weight fraction or, if nitrilotriacetate was present in the medium, some also appeared as a low molecular weight fraction. The release of non-ferritin iron was biphasic, the early, rapid phase being more prolonged with resident cells than with stimulated cells. The rate of release in the late phase did not differ significantly between resident and stimulated cells. Incubation at 0 degrees C completely suppressed the release of degraded transferrin, but iron release continued at about 30% of the rate seen in control cultures at 37 degrees C. A model for the intracellular handling of ingested iron is proposed to take account of the different release patterns of resident and stimulated macrophages.

A relationship between functional age and health status in female subjects.

A multiple regression equation was used to predict age from seven clinical variables in 1080 apparently well female subjects. A multiple correlation coefficient of R = 0.77 was achieved by five of the variables: timed forced expiratory volume, systolic blood pressure, plasma urea nitrogen, cholesterol, and alkaline phosphatase. On the basis of selection by medical questionnaire responses and other objective criteria, 9% of the subjects were nonsmokers and healthier than the rest. These selected subjects showed a significant reduction in preducted age. Within this group, subjective perception of health was associated with differences in predicted age: poor health with an increase and good health with a decrease in functional age. This study of functional age was based on the healthiest segment of the population in order to minimize the effect of overt pathological processes on the aging rate. An association has been demonstrated between health impairment and predicted age as a measure of the aging rate.

Propranolol in hypertension: a dose-response study.

The effect of propranolol was studied in a double-blind crossover trial in 24 carefully selected hypertensive outpatients. Each patient received propranolol 60 mg/day, 120 mg/day, 240 mg/day, and placebo for four weeks each according to a randomised sequence. Propranolol 60 mg/day was no better than placebo in reducing blood pressure. The effects of propranolol 120 mg/day and 240 mg/day were not significantly different. Both doses reduced lying blood pressure by about 20/10 mm Hg from an initial level of 173/104 mm Hg. No difference was detected between the effects of the different doses of propranolol and placebo on weight or on the occurrence of adverse reactions.