Pre-treatment levels of circulating free IGF-1 identify NSCLC patients who derive clinical benefit from figitumumab.

BACKGROUND: Phase III trials of the anti-insulin-like growth factor type 1 receptor (IGF-IR) antibody figitumumab (F) in unselected non-small-cell lung cancer (NSCLC) patients were recently discontinued owing to futility. Here, we investigated a role of free IGF-1 (fIGF-1) as a potential predictive biomarker of clinical benefit from F treatment. MATERIALS AND METHOD: Pre-treatment circulating levels of fIGF-1 were tested in 110 advanced NSCLC patients enrolled in a phase II study of paclitaxel and carboplatin given alone (PC) or in combination with F at doses of 10 or 20 mg kg(-1) (PCF10, PCF20). RESULTS: Cox proportional hazards model interactions were between 2.5 and 3.5 for fIGF-1 criteria in the 0.5-0.9 ng ml(-1) range. Patients above each criterion had a substantial improvement in progression-free survival on PCF20 related to PC alone. Free IGF-1 correlated inversely with IGF binding protein 1 (IGFBP-1, ρ=-0.295, P=0.005), and the pre-treatment ratio of insulin to IGFBP-1 was also predictive of F clinical benefit. In addition, fIGF-1 levels correlated with tumour vimentin expression (ρ=0.594, P=0.021) and inversely with E-cadherin (ρ=-0.389, P=0.152), suggesting a role for fIGF-1 in tumour de-differentiation. CONCLUSION: Free IGF-1 may contribute to the identification of a subset of NSCLC patients who benefit from F therapy.

The insulin-like growth factor-I receptor inhibitor figitumumab (CP-751,871) in combination with docetaxel in patients with advanced solid tumours: results of a phase Ib dose-escalation, open-label study.

BACKGROUND: This phase Ib trial assessed safety, tolerability, and maximum tolerated dose (MTD) of figitumumab (CP-751,871), a fully human monoclonal antibody targeting the insulin-like growth factor type 1 receptor (IGF-IR), in combination with docetaxel. METHODS: Patients with advanced solid tumours were treated with escalating dose levels of figitumumab plus 75 mg m(-2) docetaxel every 21 days. Safety, efficacy, pharmacokinetics (PKs), and biomarker responses were evaluated. RESULTS: In 46 patients, no dose-limiting toxicities were attributable to the treatment combination. Grade 3 and 4 toxicities included neutropaenia (n=28), febrile neutropaenia (n=11), fatigue (n=10), leukopaenia (n=7), diarrhoea (n=5), hyperglycaemia, lymphopaenia, cellulitis, DVT, and pain (all n=1). The MTD was not reached. Four partial responses were observed; 12 patients had disease stabilisation of > or =6 months. Pharmacokinetic and biomarker analyses showed a dose-dependent increase in plasma exposure, and complete sIGF-IR downregulation at doses of >or =3 mg kg(-1). Pharmacokinetics of docetaxel in combination was similar to when given alone. Out of 18 castration-resistant prostate cancer patients, 10 (56%) had > or =5 circulating tumour cells (CTCs) per 7.5 ml of blood at baseline: 6 out of 10 (60%) had a decline from > or =5 to <5 CTCs and 9 out of 10 (90%) had a > or =30% decline in CTCs after therapy. CONCLUSIONS: Figitumumab and docetaxel in combination are well tolerated. Further evaluation is warranted.

Prevention of cardiac hypertrophy in mice by calcineurin inhibition.

Hypertrophic cardiomyopathy (HCM) is an inherited form of heart disease that affects 1 in 500 individuals. Here it is shown that calcineurin, a calcium-regulated phosphatase, plays a critical role in the pathogenesis of HCM. Administration of the calcineurin inhibitors cyclosporin and FK506 prevented disease in mice that were genetically predisposed to develop HCM as a result of aberrant expression of tropomodulin, myosin light chain-2, or fetal beta-tropomyosin in the heart. Cyclosporin had a similar effect in a rat model of pressure-overload hypertrophy. These results suggest that calcineurin inhibitors merit investigation as potential therapeutics for certain forms of human heart disease.

Akt1/PKB upregulation leads to vascular smooth muscle cell hypertrophy and polyploidization.

Vascular smooth muscle cells (VSMCs) at capacitance arteries of hypertensive individuals and animals undergo marked age- and blood pressure-dependent polyploidization and hypertrophy. We show here that VSMCs at capacitance arteries of rat models of hypertension display high levels of Akt1/PKB protein and activity. Gene transfer of Akt1 to VSMCs isolated from a normotensive rat strain was sufficient to abrogate the activity of the mitotic spindle cell-cycle checkpoint, promoting polyploidization and hypertrophy. Furthermore, the hypertrophic agent angiotensin II induced VSMC polyploidization in an Akt1-dependent manner. These results demonstrate that Akt1 regulates ploidy levels in VSMCs and contributes to vascular smooth muscle polyploidization and hypertrophy during hypertension.

Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.

Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position -179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different regulatory programs in adult cardiac and skeletal muscle.

Ectopic expression of cdc2/cdc28 kinase subunit Homo sapiens 1 uncouples cyclin B metabolism from the mitotic spindle cell cycle checkpoint.

Primary human fibroblasts arrest growth in response to the inhibition of mitosis by mitotic spindle-depolymerizing drugs. We show that the mechanism of mitotic arrest is transient and implicates a decrease in the expression of cdc2/cdc28 kinase subunit Homo sapiens 1 (CKsHs1) and a delay in the metabolism of cyclin B. Primary human fibroblasts infected with a retroviral vector that drives the expression of a mutant p53 protein failed to downregulate CKsHs1 expression, degraded cyclin B despite the absence of chromosomal segregation, and underwent DNA endoreduplication. In addition, ectopic expression of CKsHs1 interfered with the control of cyclin B metabolism by the mitotic spindle cell cycle checkpoint and resulted in a higher tendency to undergo DNA endoreduplication. These results demonstrate that an altered regulation of CKsHs1 and cyclin B in cells that carry mutant p53 undermines the mitotic spindle cell cycle checkpoint and facilitates the development of aneuploidy. These data may contribute to the understanding of the origin of heteroploidy in mutant p53 cells.

Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors.

Glucocorticoids are potent immunosuppressants which work in part by inhibiting cytokine gene transcription. We show here that NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-kappa B subunits p65 and p50. In addition, we demonstrate that the ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-kappa B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-kappa B activity may be regulated by DEX. We also report that RU486 treatment of wild-type GR and DEX treatment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, we found that the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required for GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activity is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-kappa B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription.

A proliferative p53-responsive element mediates tumor necrosis factor alpha induction of the human immunodeficiency virus type 1 long terminal repeat.

Transforming mutants of the p53 tumor suppressor gene can positively regulate transcription from several promoters that do not contain known p53 binding sites. Here, we report the identification of a novel p53 binding site in the human immunodeficiency virus long terminal repeat that specifically mediates mutant p53 transactivation. This DNA element was bound by endogenous Jurkat p53 when these cells were stimulated by tumor necrosis factor. Mutation of this sequence inhibited p53 transactivation and tumor necrosis factor inducibility of the human immunodeficiency virus type 1 long terminal repeat. In addition, this DNA element was found to be sufficient to confer mutant p53 responsiveness on a heterologous minimal promoter. It has been hypothesized that transforming mutants of p53 represent a proliferative conformational stage that can be adopted by the native protein under stimulation by growth factors. The data presented suggest that proliferative and antiproliferative p53 conformations recognize different DNA binding sites in order to mediate distinct biological functions. Thus, transforming mutants of p53 that fold into the proliferative conformation would favor proliferative over antiproliferative functions.

Functional antagonism between YY1 and the serum response factor.

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.

Cyclosporin A antagonizes phenylephrine, oxytocin and angiotensin effects on glucose metabolism in rat thymus lymphocytes.

Effects of phenylephrine, oxytocin and angiotensin on fructose 2,6-bisphosphate (Fru 2,6-P2) content and glycolytic parameters were studied in incubated thymus lymphocytes. These hormones modified Fru 2,6-P2 content dependent upon the energetic status of the cells. In non-preincubated thymus lymphocytes (with relatively high levels of glycogen and ATP), phenylephrine, oxytocin and angiotensin depressed Fru 2,6-P2 content in a dose-dependent manner. The opposite was found when the cells were preincubated for 2 h without substrates (low levels of ATP and glycogen). Changes in lactate release were less evident, but significant. Phenylephrine did not modify the maximal activities of phosphofructokinase (PFK)-1 or PFK-2. However, both submaximal PFK-1 and PFK-2 activities were inhibited by phenylephrine, and the response to exogenous Fru 2,6-P2 on PFK-1 was also altered. The activities of Fru 1,6-P2 and pyruvate kinase were not modified by phenylephrine or A23187 treatment. Simultaneous presence of Cyclosporin A (CsA), an immunosuppressive drug, antagonizes the alpha-adrenergic effect on Fru 2,6-P2 content. CsA alone did not alter basal levels of ATP, hexose phosphate or Fru 2,6-P2, and its opposing effect to alpha-agonist was dose-dependent. CsA cannot change the positive action of PMA or the negative action of A23187 on Fru 2,6-P2 content. The present data suggest that CsA acts prior to calcium liberation and protein kinase C activation. Different possible molecular models are discussed.

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes.

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.

Short-term stimulation by adenosine of basal and insulin-induced glycogen synthesis in rat adipose tissue.

The effects of adenosine on glycogen metabolism have been studied in isolated fat-pads from epididymal adipose tissue. Adenosine caused a sustained short-term increase in the incorporation of [U-14C]glucose into glycogen, as well as a stimulation of both basal and insulin-induced [1-14C]glucose oxidation. Adenosine produced changes also in the activity of glycogen synthase and phosphorylase, these effects being apparent only when glucose was present in the incubation medium. The addition of adenosine prevented the depressed synthesis of glycogen observed in the presence of dibutyryl cyclic AMP. In the presence of adenosine deaminase, the stimulation by insulin of glycogen synthesis was markedly decreased. The results suggest that adenosine may have a regulatory role on glycogen synthesis by facilitating the glucose transport.

Calcineurin is activated in rat hearts with physiological left ventricular hypertrophy induced by voluntary exercise training.

BACKGROUND: Calcineurin may play a pivotal role in the signaling of cardiac hypertrophy; since this hypothesis was first put forward, controversial reports have been published using various experimental models. This study was designed to compare the physiological left ventricular hypertrophy (LVH) induced by voluntary exercise with LVH induced by aortic constriction and to determine whether calcineurin participates in the signaling of exercise-induced LVH. METHODS AND RESULTS: Wistar rats were assigned to 1 of the following 5 groups: 10 weeks of voluntary exercise (EX), a sedentary regimen, a 1-week (AC1) or 4-week (AC4) ascending aortic constriction period, or a sham operation. EX rats ran 2.4+/-0.7 km/day voluntarily in specially manufactured cages; this was associated with an increase of LV diastolic dimension and stroke volume. Myocardial calcineurin activity markedly increased in EX rats (46.4+/-8.3 versus 18.4+/-0.5 pmol. min(-1). mg(-1) in sedentary rats; P<0.001) and in AC1 rats (44.9+/-6.7 versus 22.1+/-3.7 pmol. min(-1). mg(-1) in sham-operated rats; P<0.001), but not in AC4 rats (29.0+/-3.4 pmol. min(-1). mg(-1)). Treatment with cyclosporin A completely inhibited the development of LVH in EX rats, but it only partially attenuated the development of LVH in AC4 rats. CONCLUSIONS: Calcineurin was activated in exercise-induced physiological LVH and in the developing phase of LVH (AC1), but not in decompensated pressure-overload hypertrophy (AC4). Cyclosporin therapy for the prevention of LVH may be harmful because it does not block the development of pathological hypertrophy but rather that of favorable adaptive hypertrophy.

The gene encoding rat phosphoglycerate mutase subunit M: cloning and promoter analysis in skeletal muscle cells.

The expression of the gene encoding the muscle-specific (M)-subunit of phosphoglycerate mutase (PGAM-M) is restricted to adult skeletal and cardiac muscle. In order to study its expression in muscle, the rat PGAM-M gene has been isolated and sequenced. Rat PGAM-M spans about 2.2 kb and is composed of three exons: 442, 181 and 186-bp long, and two introns of 97 bp and 1.3 bp. The analysis of the 5'-flanking region reveals a promoter which contains multiple DNA regulatory elements and constitutes an ideal model to study muscle gene transcriptional regulation. Thus, the elements responsible for rat PGAM-M muscle-specific expression have been identified by transient transfection in chicken embryo primary cultures, using chimeric constructs of the rat promoter linked to a cat reporter gene. Here, we report that in spite of the abundance of E-box motifs in the rat PGAM-M promoter known for their involvement in muscle gene expression, two DNA elements regulate the muscle-specific transcription of rat PGAM-M: an A/T motif, the putative MEF-2-binding site (myocyte-specific enhancer-binding factor 2), and a proximal 27-bp element which is conserved between the rat and human genes. These two elements define a small promoter (170 bp) sufficient to support potent and skeletal-muscle-specific expression. The conserved 27-bp region contains a transcriptional regulatory element able to confer muscle-specific expression when located upstream from a heterologous TATA box.

Hormonal regulation of fructose 2,6-bisphosphate levels in epididymal adipose tissue of rat.

The participation of fructose 2,6-bisphosphate on glycolysis stimulated by insulin and adrenaline in incubated white adipose tissue of rat was investigated. Adrenaline addition to incubated fat-pads strongly decreased the intracellular levels of fructose 2,6-bisphosphate. When the tissue was preincubated with glucose, the presence of insulin in the incubation medium increased fructose 2,6-bisphosphate levels 2-fold. These variations were related to changes in the substrates, ATP and fructose 6-phosphate. It therefore appears that fructose 2,6-bisphosphate may be involved in the control of insulin-induced glycolysis, but it does not seem to play a role in the stimulation of glucolysis by adrenaline.

Regulation of fructose 2,6-bisphosphate levels in cold-acclimated brown adipose tissue of rat.

The effects of cold exposure and T4 administration on fructose 2,6-bisphosphate levels, phosphofructokinase-2 and pyruvate kinase activities were examined in rat brown adipose tissue. Cold adaptation (14 days) gave rise to a 2-fold increase in the amount of fructose 2,6-bisphosphate and phosphofructokinase-2 activity, and increased the pyruvate kinase activity 4-fold. If, in addition, the cold-acclimated rats were treated with T4, these parameters were again significantly enhanced. The effect on phosphofructokinase-2 was on the Vmax, without modification of the Km (for both fructose 6-phosphate and ATP) of the enzyme. In the hypothyroid state, however, the activity of pyruvate kinase remains unchanged. These data support previous observations on stimulation of glycolytic flux during cold adaptation in brown adipose tissue, and a permissive role of thyroid hormones in the process.

The control of mitosis.

A precise coordination of multiple cell cycle events is required to ensure proper mitosis. Chromosome cohesion must be maintained until all chromosomes are attached to opposite poles of the mitotic spindle and aligned at the metaphase plate. At the onset of anaphase, the activity of separins contributes to the release of cohesins from chromosomes, allowing for the segregation of bivalents to opposite spindle poles. Separin activity is blocked by binding to a class of proteins known as securins, whose turnover at the metaphase-to-anaphase transition is triggered by the Anaphase Promoting Complex or cyclosome. The mitotic spindle cell cycle checkpoint coordinates the timing of these events and acts as input mechanism for DNA damage/stress pathways. Failure of this precise network leads to genomic instability and/or cell death.

Gain of function properties of mutant p53 proteins at the mitotic spindle cell cycle checkpoint.

Mutations in the p53 tumor suppressor gene locus predispose human cells to chromosomal instability. This is due in part to interference of mutant p53 proteins with the activity of the mitotic spindle and postmitotic cell cycle checkpoints. Recent data demonstrates that wild type p53 is required for postmitotic checkpoint activity, but plays no role at the mitotic spindle checkpoint. Likewise, structural dominant p53 mutants demonstrate gain-of-function properties at the mitotic spindle checkpoint and dominant negative properties at the postmitotic checkpoint. At mitosis, mutant p53 proteins interfere with the control of the metaphase-to-anaphase progression by up-regulating the expression of CKs1, a protein that mediates activatory phosphorylation of the anaphase promoting complex (APC) by Cdc2. Cells that carry mutant p53 proteins overexpress CKs1 and are unable to sustain APC inactivation and mitotic arrest. Thus, mutant p53 gain-of-function at mitosis constitutes a key component to the origin of chromosomal instability in mutant p53 cells.

A competitive mechanism of CArG element regulation by YY1 and SRF: implications for assessment of Phox1/MHox transcription factor interactions at CArG elements.

In the promoters of many immediate early genes, including c-fos, CArG DNA regulatory elements mediate basal constituitive expression and rapid and transient serum induction. CArG boxes also occur in the promoters of muscle-specific genes, including skeletal alpha-actin, where it confers muscle-specific expression. These elements are regulated, at least in part, by the ubiquitous transcription factors serum response factor (SRF) and YY1. The homeobox transcription factor Phox1/MHox has also been implicated in regulation of the c-fos CArG element and is thought to function by facilitating SRF binding to DNA. Here, we provide in vitro and in vivo evidence that the mechanism of YY1 repression of CArG elements results from competition with SRF for overlapping binding sites. We describe in detail the binding sites of YY1 and SRF through serial point mutations of the skeletal alpha-actin proximal CArG element and identify a mutation that dramatically reduces YY1 binding but retains normal SRF binding. YY1 competes with SRF for binding to wild-type CArG elements, but not to this point mutant in vitro. This mutant is sufficient for muscle-specific expression in vivo but is much less sensitive to repression by YY1 overexpression. We utilized the YY1/SRF competition to address the role of Phox1 at these elements. Phox1 overexpression did not diminish YY1-mediated repression, suggesting that transcriptional activation by Phox1 does not result from enhanced SRF binding to these elements. These methods may prove to be useful for assessing interactions between other CArG element regulatory factors.