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Objective: In order to search for metabolic biomarkers of antihypertensive drug responsiveness, we measured >600 biochemicals in plasma samples of subjects participating in the GENRES Study. Hypertensive men received in a double-blind rotational fashion amlodipine, bisoprolol, hydrochlorothiazide and losartan, each as a monotherapy for one month, with intervening one-month placebo cycles., Methods: Metabolomic analysis was carried out using ultra high performance liquid chromatography-tandem mass spectrometry. Full metabolomic signatures (the drug cycles and the mean of the 3 placebo cycles) became available in 38 to 42 patients for each drug. Blood pressure was monitored by 24-h recordings., Results: Amlodipine (P values down to 0.002), bisoprolol (P values down to 2 x 10-5) and losartan (P values down to 2 x 10-4) consistently decreased the circulating levels of long-chain acylcarnitines. Bisoprolol tended to decrease (P values down to 0.002) the levels of several medium- and long-chain fatty acids. Hydrochlorothiazide administration was associated with an increase of plasma uric acid level (P = 5 x 10-4) and urea cycle metabolites. Decreases of both systolic (P = 0.06) and diastolic (P = 0.04) blood pressure after amlodipine administration tended to associate with a decrease of plasma hexadecanedioate, a dicarboxylic fatty acid recently linked to blood pressure regulation., Conclusions: Although this systematic metabolomics study failed to identify circulating metabolites convincingly predicting favorable antihypertensive response to four different drug classes, it provided accumulating evidence linking fatty acid metabolism to human hypertension.

Aim: To replicate the genome-wide associations of the antihypertensive effects of bisoprolol and losartan in GENRES, using the Finnish patients of LIFE study., Patients & Methods: We analyzed association of four SNPs with atenolol and three SNPs with losartan response in 927 Finnish LIFE patients (467 for atenolol and 460 for losartan)., Results: rs2514036, a variation at a transcription start site of ACY3, was associated with blood pressure response to atenolol in men in LIFE. Response to bisoprolol was correlated to baseline plasma levels of N-acetylphenylalanine and phenylalanine (ACY3 substrate and end product, respectively) in GENRES study. NPHS1 variation rs3814995 was associated with losartan effect in LIFE., Conclusion: We provide support for two pharmacogenomic markers for beta-blockers and angiotensin receptor antagonists.

Introduction: Freeze-dried black raspberries (BRBs) elicit chemopreventive effects against colorectal cancer in humans and in rodents. The objective of this study was to investigate potential BRB-caused metabolite changes using wild-type (WT) C57BL/6 mice., Methods and Results: WT mice were fed either control diet or control diet supplemented with 5% BRBs for 8 wk. A nontargeted metabolomic analysis was conducted on colonic mucosa, liver, and fecal specimens collected from both diet groups. BRBs significantly changed the levels of 41 colonic mucosa metabolites, 40 liver metabolites, and 34 fecal metabolites compared to control diet-fed mice. BRBs reduced 34 lipid metabolites in colonic mucosa and increased levels of amino acids in liver. One metabolite, 3-[3-(sulfooxy) phenyl] propanoic acid, might be a useful biomarker of BRB consumption. In addition, BRB powder was found to contain 30-fold higher levels of linolenate compared to control diets. Consistently, multiple omega-3 polyunsaturated fatty acids (ω-3 PUFAs), including stearidonate, docosapentaenoate (ω-3 DPA), eicosapentaenoate (EPA), and docosahexaenoate (DHA), were significantly elevated in livers of BRB-fed mice., Conclusion: The data from the current study suggest that BRBs produce systemic metabolite changes in multiple tissue matrices, supporting our hypothesis that BRBs may serve as both a chemopreventive agent and a beneficial dietary supplement.

Dysregulation of receptor tyrosine kinases (RTK) contributes to cellular transformation and cancer progression by disrupting key metabolic signaling pathways. The EPHA2 RTK is overexpressed in aggressive forms of breast cancer, including the HER2(+) subtype, and correlates with poor prognosis. However, the role of EPHA2 in tumor metabolism remains unexplored. In this study, we used in vivo and in vitro models of HER2-overexpressing breast cancer to investigate the mechanisms by which EPHA2 ligand-independent signaling promotes tumorigenesis in the absence of its prototypic ligand, ephrin-A1. We demonstrate that ephrin-A1 loss leads to upregulated glutamine metabolism and lipid accumulation that enhanced tumor growth. Global metabolic profiling of ephrin-A1-null, HER2-overexpressing mammary tumors revealed a significant increase in glutaminolysis, a critical metabolic pathway that generates intermediates for lipogenesis. Pharmacologic inhibition of glutaminase activity reduced tumor growth in both ephrin-A1-depleted and EPHA2-overexpressing tumor allografts in vivo Mechanistically, we show that the enhanced proliferation and glutaminolysis in the absence of ephrin-A1 were attributed to increased RhoA-dependent glutaminase activity. EPHA2 depletion or pharmacologic inhibition of Rho, glutaminase, or fatty acid synthase abrogated the increased lipid content and proliferative effects of ephrin-A1 knockdown. Together, these findings highlight a novel, unsuspected connection between the EPHA2/ephrin-A1 signaling axis and tumor metabolism, and suggest potential new therapeutic targets in cancer subtypes exhibiting glutamine dependency. Cancer Res; 76(7); 1825-36. ©2016 AACR., (©2016 American Association for Cancer Research.)

Dysregulated metabolism is a hallmark of cancer, manifested through alterations in metabolites. We performed metabolomic profiling on 138 matched clear cell renal cell carcinoma (ccRCC)/normal tissue pairs and found that ccRCC is characterized by broad shifts in central carbon metabolism, one-carbon metabolism, and antioxidant response. Tumor progression and metastasis were associated with metabolite increases in glutathione and cysteine/methionine metabolism pathways. We develop an analytic pipeline and visualization tool (metabolograms) to bridge the gap between TCGA transcriptomic profiling and our metabolomic data, which enables us to assemble an integrated pathway-level metabolic atlas and to demonstrate discordance between transcriptome and metabolome. Lastly, expression profiling was performed on a high-glutathione cluster, which corresponds to a poor-survival subgroup in the ccRCC TCGA cohort., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Freeze-dried black raspberries (BRBs) have demonstrated chemopreventive effects in a dietary intervention trial with human colorectal cancer patients. The aim of this study was to investigate BRB-caused metabolite changes using the Apc(Min/+) mouse as a model of human colorectal cancer. Wild-type (WT) mice were fed control diet, and Apc(Min/+) mice were fed either control diet or control diet supplemented with 5% BRBs for 8 weeks. Colonic and intestinal polyp size and number were measured. A non-targeted metabolomic analysis was conducted on colonic mucosa, liver and fecal specimens. Eight weeks of BRB treatment significantly decreased intestinal and colonic polyp number and size in Apc(Min/+) mice. The apc gene mutation significantly changed 52 metabolites in colonic mucosa associated with increased amino acid and decreased lipid metabolites, as well as 39 liver and 8 fecal metabolites. BRBs significantly reversed 23 apc-regulated metabolites, including 13 colonic mucosa, 8 liver and 2 fecal metabolites that were involved in amino acid, glutathione, lipid and nucleotide metabolism. Of these, changes in eight metabolites were linearly correlated with decreased colonic polyp number and size in BRB-treated Apc(Min/+) mice. Elevated levels of putrescine and linolenate in Apc(Min/+) mice were significantly decreased by BRBs. Ornithine decarboxylase expression, the key enzyme in putrescine generation, was fully suppressed by BRBs. These results suggest that BRBs produced beneficial effects against colonic adenoma development in Apc(Min/+) mice and modulated multiple metabolic pathways. The metabolite changes produced by BRBs might potentially reflect the BRB-mediated chemopreventive effects in colorectal cancer patients., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Understanding the specific survival of the rare chronic myelogenous leukaemia (CML) stem cell population could provide a target for therapeutics aimed at eradicating these cells. However, little is known about how survival signalling is regulated in CML stem cells. In this study, we survey global metabolic differences between murine normal haematopoietic stem cells (HSCs) and CML stem cells using metabolomics techniques. Strikingly, we show that CML stem cells accumulate significantly higher levels of certain dipeptide species than normal HSCs. Once internalized, these dipeptide species activate amino-acid signalling via a pathway involving p38MAPK and the stemness transcription factor Smad3, which promotes CML stem cell maintenance. Importantly, pharmacological inhibition of dipeptide uptake inhibits CML stem cell activity in vivo. Our results demonstrate that dipeptide species support CML stem cell maintenance by activating p38MAPK-Smad3 signalling in vivo, and thus point towards a potential therapeutic target for CML treatment.

Dietary intervention of freeze-dried black raspberries (BRBs) in a group of human colorectal cancer patients has demonstrated beneficial effects, including proapoptosis, antiproliferation, and antiangiogenesis. The aim of this study was to investigate BRB-mediated metabolite changes from this same cohort of patients. Twenty-eight colorectal cancer patients were given 60 g BRB powder daily for 1 to 9 weeks. Urine and plasma specimens were collected before and after BRB intervention. A mass spectrometry-based nontargeted metabolomic analysis was conducted on each specimen. A total of more than 400 metabolites were annotated in each specimen. Of these 34 and 6 metabolites were significantly changed by BRBs in urine and plasma, respectively. Increased levels of 4-methylcatechol sulfate in both post-BRB urine and post-BRB plasma were significantly correlated with a higher level of apoptotic marker (TUNEL) in post-BRB tumors. One tricarboxylic acid (TCA) cycle metabolites, cis-aconitate, was increased in post-BRB urine. Furthermore, BRB-derived polyphenols were absorbed and metabolized to various benzoate species, which were significantly increased in post-BRB specimens. Increased benzoate levels were positively correlated with enhanced levels of amino acid metabolite. These results suggest that BRBs induce significant metabolic changes and affect energy generating pathways.This study supports the hypothesis that BRBs might be beneficial to colorectal cancer patients through the regulation of multiple metabolites., (©2015 American Association for Cancer Research.)

Introduction: There is a critical need to develop clinical laboratory assays that provide risk assessment for men at elevated risk for prostate cancer, and once diagnosed, could further identify those men with clinically significant disease., Methods: Recent advancements in analytical instrumentation have enabled mass spectrometry-based metabolomics methodologies. Further advancements in chromatographic techniques have facilitated high throughput, quantitative assays for a broad spectrum of biochemicals., Results: Screening metabolomics techniques have been applied to biospecimens from large cohorts of men comparing those individuals with prostate cancer to those with no evidence of malignancy. Work beginning in tissues has identified biochemical profiles that correlate with disease and disease severity, including tumor grade and stage. Some of these metabolic abnormalities, such as dramatic elevations in sarcosine, have been found to translate into biological fluids, especially blood and urine, which can be sampled in a minimally invasive manner., Discussion: The differential abundances of these tumor-associated metabolites have been found to improve the performance of clinical prognostic/diagnostic tools., Conclusion: The outlook is bright for metabolomic technology to address clinical diagnostic needs for prostate cancer patient management. Early validation of specific clinical tests provides a preview of further successes in this area. Metabolomics has shown its utility to complement and augment traditional clinical approaches as well as emerging genomic, transcriptomic and proteomic methodologies, and is expected to play a key role in the precision medicine-based management of the prostate cancer patient.

Bladder cancer (BCa) is a common malignancy worldwide and has a high probability of recurrence after initial diagnosis and treatment. As a result, recurrent surveillance, primarily involving repeated cystoscopies, is a critical component of post diagnosis patient management. Since cystoscopy is invasive, expensive and a possible deterrent to patient compliance with regular follow-up screening, new non-invasive technologies to aid in the detection of recurrent and/or primary bladder cancer are strongly needed. In this study, mass spectrometry based metabolomics was employed to identify biochemical signatures in human urine that differentiate bladder cancer from non-cancer controls. Over 1000 distinct compounds were measured including 587 named compounds of known chemical identity. Initial biomarker identification was conducted using a 332 subject sample set of retrospective urine samples (cohort 1), which included 66 BCa positive samples. A set of 25 candidate biomarkers was selected based on statistical significance, fold difference and metabolic pathway coverage. The 25 candidate biomarkers were tested against an independent urine sample set (cohort 2) using random forest analysis, with palmitoyl sphingomyelin, lactate, adenosine and succinate providing the strongest predictive power for differentiating cohort 2 cancer from non-cancer urines. Cohort 2 metabolite profiling revealed additional metabolites, including arachidonate, that were higher in cohort 2 cancer vs. non-cancer controls, but were below quantitation limits in the cohort 1 profiling. Metabolites related to lipid metabolism may be especially interesting biomarkers. The results suggest that urine metabolites may provide a much needed non-invasive adjunct diagnostic to cystoscopy for detection of bladder cancer and recurrent disease management.

Lipid nanoparticles (LNPs) represent the most clinically advanced technology for the systemic delivery of therapeutic siRNA in vivo. Toward this end, a novel class of LNPs comprising low molecular weight (MW) ionizable amino lipids having asymmetric architecture was recently reported.1 LNPs of these amino lipids, termed asymmetric LNPs, were shown to be highly efficacious and well tolerated in vivo; advances were enabled by improved endosomal escape, coupled with enhanced amino lipid metabolism and clearance. In this work, we show that, in contrast to their desirable pharmacological performance, asymmetric LNPs present a significant pharmaceutical developability challenge, namely physical instability limiting extended shelf life. Using orthogonal characterization methods, we identify the mechanism of LNP instability as Ostwald ripening and establish it to be driven predominantly by the asymmetric amino lipid component. Through rational optimization of LNP physical and macromolecular properties, we are able to significantly attenuate or entirely eliminate the Ostwald ripening instability. Modulation of LNP size, for example, effectively halts particle growth. Similarly, optimization of LNP macromolecular packing through deliberate selection of structurally matched colipids significantly diminishes the rate of ripening. This later experimental observation is substantiated by molecular dynamics simulations of LNP self-assembly, which establish a quantitative dependence of LNP macromolecular order on colipid structure. In totality, the experimental and molecular dynamics outcomes of this work support the rational design of LNP physical and chemical properties leading to effective Ostwald ripening stabilization and enable the advance of asymmetric LNPs as a clinic-ready platform for siRNA therapeutics.

Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.

The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC(50)=3.1 nM).

The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.

The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.

The x-ray structure of the unactivated kinase domain of insulin-like growth factor-1 receptor (IGFRK-0P) is reported here at 2.7 A resolution. IGFRK-0P is composed of two lobes connected by a hinge region. The N-terminal lobe of the kinase is a twisted beta-sheet flanked by a single helix, and the C-terminal lobe comprises eight alpha-helices and four short beta-strands. The ATP binding pocket and the catalytic center reside at the interface of the two lobes. Despite the overall similarity to other receptor tyrosine kinases, three notable conformational modifications are observed: 1) this kinase adopts a more closed structure, with its two lobes rotated further toward each other; 2) the conformation of the proximal end of the activation loop (residues 1121-1129) is different; 3) the orientation of the nucleotide-binding loop is altered. Collectively, these alterations lead to a different ATP-binding pocket that might impact on inhibitor designs for IGFRK-0P. Two molecules of IGFRK-0P are seen in the asymmetric unit; they are associated as a dimer with their ATP binding clefts facing each other. The ordered N terminus of one monomer approaches the active site of the other, suggesting that the juxtamembrane region of one molecule could come into close proximity to the active site of the other.

Activation of phosphoinositide 3'-hydroxykinase (P13K) is required for mitogenic signal transduction by several growth factors and oncogenes. P13K is a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit. In the current study, we report the cloning and characterization of the p110 alpha catalytic subunit of human P13K. This clone is highly homologous (> 99% amino acid identity) to bovine brain p110 alpha, but contains 10 amino acid differences from the human p110 alpha sequence previously reported. Comparison of this sequence with known Ser/Thr kinases and p110 homologs highlighted several conserved residues within the putative kinase domain. Mutational analysis of these residues (Asp915, (Asp933 + Phe934)) yielded P13K mutants with virtually complete loss of phosphoinositide phosphorylating activity. Expression of the wild-type p110 alpha protein in CHO cells is sufficient to activate the serum response element derived from the promoter of c-fos, an immediate early gene product. In contrast, the catalytically impaired p110 alpha mutants as well as the p85 alpha subunit of P13K were inactive in the fos assay. These studies suggest that the mitogenic signal transduction pathway mediated by P13K is dependent upon the enzymatic activity of the p110 alpha subunit of P13K.

Phosphorylation of phosphoinositides by phosphoinositide 3'-hydroxykinase (PI3K) occurs at a lipid/water interface. We have determined that highly purified recombinant human P13K binds tightly to vesicle interfaces composed primarily of phosphatidylinositol (PI) or 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM). The rate of desorption of PI3K from the vesicle interface is slow and does not significantly affect the observed product formation kinetics. Observations which demonstrate that PI3K is tightly bound to the vesicle lipid/water interface include the following: (1) product formation plateaus rapidly, even in the presence of active enzyme and excess substrate; (2) total product formation is proportional to the amount of PI3K; (3) initial product formation rates are unaffected by bulk lipid concentration but are dependent on the interfacial substrate concentration; and (4) PI3K partitions with lipid vesicles in sedimentation gradients. This enzymatic profile has been referred to as catalysis in the "scooting" mode (Berg et al., 1991). A kinetic analysis of PI3K catalysis in the scooting mode is presented. The interfacial Km,app for PI was determined to be approximately 6.0 mol % in PI/DMPM vesicles. The ratio of specificity constants (kcat/Km) for PI, phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-diphosphate (PIP2) utilization was determined to be near unity. These results provide a rigorous enzymological framework for the kinetic analysis of PI3K inhibitors.

The retinoblastoma gene product (pRB) participates in regulating mammalian cell replication. The mechanism responsible for pRB's growth regulatory activity is uncertain. However, pRB is known to bind viral transforming proteins including the papilloma virus E7 protein, cellular proteins, and DNA. pRB contains a critical domain termed the "binding pocket" which is required for binding activities. This binding pocket contains 8 cysteine residues. A naturally occurring mutation affecting one of these cysteines is known to eliminate pRB's protein and DNA binding activities. To investigate the cysteine residues in pRB's binding pocket, each residue was mutated to alanine, phenylalanine, or serine. These mutant genes were used to prepare pRBs harboring specific amino acid substitutions. Individual mutations at positions 407, 553, 666, and 706 depressed pRB binding to E7 protein, DNA, and a conformation-specific anti-pRB antibody, XZ133. Combinations of these inhibitory mutations exhibited additive inhibitory effects on pRB's binding properties. Mutations at positions 438, 489, 590, 712, and 853 did not affect pRB binding to E7 protein, DNA, or the XZ133 antibody. Combination of these five neutral mutations yielded a pRB species with full E7 protein, DNA, and XZ133 binding activities. These studies indicate that the cysteine residues at positions 407, 553, 666, and 706 contribute to the E7 protein and DNA binding properties of pRB and appear to do so by maintaining pRB's normal conformation.

Physiological effects of isopropyl-thiogalactopyranoside (IPTG) induction were examined in Escherichia coli strain JM109 expressing a fusion protein composed of transforming growth factor alpha and a 40-kD portion of Pseudomonas aeruginosa exotoxin A (TGF(alpha)-PE40) under control of the tac promoter. Fermentations at the 15-L scale in complex medium compared growth and metabolite profiles of the untransformed JM109 host strain, the strain transformed with the vector lacking the TGF(alpha)-PE40 open reading frame (JM109[pKK2.7]), and the strain with the complete plasmid for TGF(alpha)-PE40 expression (JM109[pTAC-TGF57-PE40]). Metabolite and growth profiles of JM109 (pTAC-TGF57-PE40) cultures changed significantly in IPTG-induced versus uninduced cultures. Prior to induction, glucose was metabolized to acetate or completely oxidized to CO(2). Following induction, pyruvate was also excreted in addition to acetate. In the absence of inducer, pyruvate was excreted by JM109 (pTAC-TGF57-PE40) only when dissolved oxygen levels fell to less than 10% of saturation (microaerobic rather than anaerobic conditions). The untransformed JM109 host strain or JM109 (pKK2.7) did not excrete pyruvate in the presence or absence of inducer, although JM109 (pKK2.7) exhibited a pattern of growth following addition of IPTG that closely resembled JM109 (pTAC-TFG57-PE40). Fermentations of JM109 (pTAC-TFG57-PE40) in a synthetic medium supported lower expression levels, but resulted in similar alterations in metabolite profiles. Induction in synthetic medium resulted in pyruvate excretion without further acetate accumulation. Taken together, these data suggest that one consequence of TGF(alpha)-PE40 expression in JM109 is altered patterns of pyruvate oxidation., ((c) 1992 John Wiley & Sons, Inc.)

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.

Transforming growth factor-alpha-Pseudomonas exotoxin-40 (TP40) is a recombinant fusion protein. TP40 consists of the entire human transforming growth factor-alpha (TGF alpha) protein fused to a 40,000 Da. segment of the Pseudomonas exotoxin A protein. TP40 is a bifunctional molecule that possesses the epidermal growth factor (EGF) receptor binding properties of TGF alpha and the cell killing properties of Pseudomonas exotoxin A. These properties make TP40 a selective cytotoxic agent that kills EGF receptor bearing cells. TP40 has been shown to effectively kill human tumor cell lines that possess EGF receptors in vitro and in nude mice. In the present study, TP40 was tested against tumors taken directly from patients and grown in a soft agar human tumor cloning system. A total of 107 patients' tumors (taken from patients with tumors refractory to chemotherapy) were tested with a continuous exposure to 0.5-50 nM concentrations of the agent. TP40 exhibited a clear dose response effect against a wide variety of human solid tumor colony-forming units with greater than or equal to 84% of evaluable tumors responding at a drug concentration greater than or equal to 24 nM. When used as a continuous exposure, concentrations of TP40 as low as 5 nM demonstrated substantial in vitro activity. This activity included cytotoxicity against breast, colorectal, endometrial, head and neck, non small-cell lung, gastric, sarcoma, and pancreatic cancer tumor colony-forming units. Additional in vivo testing of this compound is warranted.

Transforming growth factor-alpha (TGF alpha)-pseudomonas exotoxin-40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the pseudomonas exotoxin A protein. TGF alpha-PE40 exhibits the receptor binding activity of TGF alpha and the cell killing activity of PE40. In the current study, we report that a modified TGF alpha-PE40 derivative significantly prolongs the survival of nude mice bearing tumors derived from cell lines which express the epidermal growth factor receptor (EGFR). In addition, the therapeutic benefit of this protein is mediated by specific binding to the EGF receptor. These results indicate that a therapeutic window exists in vivo for the use of some growth factor--toxin fusion proteins as anticancer agents.

Transforming growth factor alpha (TGF alpha)-Pseudomonas exotoxin 40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the Pseudomonas exotoxin A protein. TGF alpha-PE40 exhibits the receptor-binding activity of TGF alpha and the cell-killing activity of PE40. These properties make TGF alpha-PE40 an effective cytotoxic agent for cells that possess epidermal growth factor receptors (EGFR). However, the utility of this protein as an anticancer agent has been unclear because many normal tissues express EGFR and may be damaged by exposure to TGF alpha-PE40. To address this issue, we injected nude mice with a lethal inoculum of either A431 or HT29 human tumor cells that possess EGFR or with Chinese hamster ovary (CHO) tumor cells that lack EGFR. Animals were treated with a derivative of TGF alpha-PE40 in which the cysteine residues are replaced by alanine, termed "TGF alpha-PE40 delta cys," or with saline once a day for 5 days. Mice bearing EGFR+ tumor cells lived significantly (P less than 0.001) longer when treated with TGF alpha-PE40 delta cys compared with saline-treated controls (median survival: A431 cells, 51.5 vs. 25.5 days; HT29 cells, 101 vs. 47.5 days). TGF alpha-PE40 delta cys did not prolong the survival of mice bearing tumor cells that lack EGFR (median survival: CHO cells, 15.5 vs. 19.5 days). The only toxicity to normal tissues was mild periportal hepatic necrosis. These studies indicate that a therapeutic window exists in vivo for the use of some growth factor-toxin fusion proteins as anticancer agents.

We have evaluated the B to Z conformational transitions in supercoiled recombinant plasmids containing different lengths of (dC-dG) described in the preceding paper. The sodium chloride-induced right- to left-handed transition in a small segment of the plasmids caused a relaxation of (-) supercoils which was monitored by electrophoretic mobility changes of individual topoisomers on agarose gels containing NaCl at concentrations up to 5.0 M. The number of supercoils relaxed was proportional to the length of the (dC-dG) segment in the plasmid in good agreement with theoretical values. A short B/Z junction region (less than 5 base pairs) was inferred. The stability of the Z conformation in (dC-dG) segments of the plasmids had a strong length dependency; shorter lengths were less stable. Ten base pairs of (dC-dG) was insufficient to allow a Z conformation under the conditions studied. Supercoiling imparts a substantial favorable free energy to the Z conformation, reducing the NaCl concentration necessary to cause the transition. The relationship of supercoiling with the NaCl concentration necessary to cause a B leads to Z transition suggests that supercoiling alone is sufficient to stabilize the Z conformation at physiological salt concentrations. These results support the notion that left-handed DNA has an important biological role.

CD spectroscopy on the double-stranded strictly alternating dinucleotide polymer (dT-dG)n . (dC-dA)n partially modified by N-acetoxy-N-acetyl-2-aminofluorene suggests a left-handed conformation in concentrated NaCl solutions. Modification of the (dT-dG)n . (dC-dA)n polymer with acetylaminofluorene is required to promote formation of the left-handed helix since high salt concentrations and several other ionic conditions, which cause a similar transition for (dG-dC)n . (dG-dC)n, are ineffective. Furthermore, substitution of dC with 5-methyl dC in (dT-dG)n . (dC-dA)n does not facilitate formation of a left-handed helix, also in contrast to results found for (dG-dC)n . (dG-dC)n. A 62-base pair tract of almost perfectly alternating (dT-dG)n . (dC-dA)n from the 3'-side of the mouse kappa immunoglobulin gene modified with acetylaminofluorene undergoes the salt-induced transition to a left-handed helix when studied within a 140-base pair restriction fragment. High NaCl concentrations alone will not cause the transition for this 62-base pair tract in this fragment nor in the recombinant plasmid pRW777, which contains this fragment.

Recombinant pBR322 derivatives were constructed containing tracts of (dC-dG) sequences which are 58, 32, 26, and 10 base pairs (bp) in length in conjunction with a 95-bp fragment containing the Escherichia coli lac operator-promoter. The biological properties of these plasmids were unusual since deletions in the (dC-dG) regions, but not in the pBR322 nor the lac segments, were frequently observed; segments of (dC-dG) longer than approximately 50 bp were not stable but suffered deletions. Segments of approximately 30 bp or shorter were stable in most cases. The (dC-dG) tracts seemed to enhance recA-mediated recombination when they were of suitable length and were cloned into certain sites in the recombinants. Also the (dC-dG)-containing plasmids were less supercoiled (by 6-12 turns) than expected, relative to control plasmids, after isolation from E. coli hosts. These recombinant plasmids were used in the following paper to evaluate the properties of segments containing the unorthodox left-handed conformations.

In physiological ionic conditions (200 mM NaCl), the (dC-dG)16 and (dC-dG)13 blocks in plasmid pRW751 are in a left-handed state when the negative superhelical density of the plasmid is greater than 0.972. As the salt concentration decreases or when (dmC-dG) sequences are present, less negative supercoiling is required to induce the right- to left-handed DNA transition. Furthermore, the single strand-specific nuclease, S1, recognizes and cleaves aberrant structural features at the junction between neighbouring right- and left-handed DNA regions.

Circular dichroism and 31P-NMR on synthetic oligomers of (dC-dG) inserted within DNA restriction fragments indicate that the right-handed B-structure can exist in close proximity to the left-handed Z-structure. Also, this salt-induced transition to Z-form in a small (dC-dG) segment (1.3%) of a recombinant plasmid markedly influenced the supercoil of the plasmid. These observations have implications for the postulated role of naturally occurring related simple sequences in the regulation of gene activity.

Two adjacent, divergently transcribed, developmentally regulated genes of the maize chloroplast chromosome have different superhelical density/transcriptional activation profiles when transcribed in vitro by the homologous DNA-dependent RNA polymerase. Promoter-specific transcription of the gene for the beta and epsilon subunits of coupling factor 1 (cf1BE) increases and plateaus from templates of increasing negative superhelicity, while transcription of the gene for ribulose bisphosphate carboxylase large subunit (rcL) rises and then falls. Maximal transcription from the two promoters occurs at different template negative superhelical densities and transcription of the two genes is stimulated to different degrees. The different superhelicity profiles alter the molar ratios of the two transcripts over an order of magnitude. Changes in DNA conformation represent one possible mechanism for the differential regulation of the genes.

Studies on DNA polymers, restriction fragments, and recombinant plasmids have revealed the following: A) A family of left-handed DNA conformations exists for (dC-dG)n.(dC-dG)n. The observation of a particular conformation is dependent on the salt, the salt concentration and dehydrating agent. B) In sodium acetate solutions, (dC-dG)n.(dC-dG)n forms left-handed, psi(+)-condensed structures as detected by Raman spectroscopy and circular dichroism. C) (dT-dG)n.(dC-dA)n undergoes a right-to-left-handed transition only when reacted with AAF and at high salt concentrations. D) Transitions observed for polymer DNAs also are observed for restriction fragments containing both (dC-dG).(dC-dG) and (dT-dG).(dC-dA) sequences, but the transitions in the fragments generally require higher salt concentrations than observed for the polymers. E) Studies with recombinant plasmids containing (dC-dG) sequences from 10 to 58 bp in length demonstrate that left-handed Z-DNA segments can exist contiguous to B-DNA segments. F) Negative supercoil density (sigma less than or equal to -0.072) is sufficient to convert the (dC-dG) regions in those plasmids into left-handed structures under physiological ionic conditions (200 mM NaCl). G) The favorable free energy contribution of methylation in stabilizing the Z form in fragments and plasmids is approximately offset by the unfavorable free energy contributions of the B/Z junctions. H) Sl and BAL 31 nucleases recognize aberrant structural features at the confluence of the B and Z regions. I) Detailed mapping of Sl nuclease cleavage on supercoiled plasmids shows that the nuclease sensitive regions extend over at least five to ten bp. J) Even though the (dT-dG)n.(dC-dA)n polymer requires base modification and high salt conditions to undergo the R----L transition, supercoiling (sigma less than or equal to -0.07) can supply enough energy to allow a plasmid containing the intervening sequence of a human fetal globin gene with (dT-dG).(dC-dA) sequences to undergo a R----L transition.

Alternating (dC-dG)n regions in DNA restriction fragments and recombinant plasmids were methylated at the 5 position of the cytosine residues by the HhaI methylase. Methylation lowers the concentration of NaCl or MgCl2 necessary to cause the B-Z conformational transition in these sequences. Ionic strengths higher than physiological conditions are required to form the Z conformation when the methylated (dC-dG)n tract is contiguous with regions that do not form Z structures, in contrast to the results with the DNA polymer poly(m5dC-dG) . poly(m5dC-dG). In supercoiled plasmids containing (dC-dG)n sequences, methylation reduces the number of negative supercoils necessary to stabilize the Z conformation. Calculations of the observed free energy contributions of the B-Z junction and cytosine methylation suggest that two junctions offset the favorable effect of methylation on the Z conformation in (dC-dG)n sequences (about 29 base-pairs in length). Studies with individual methylated topoisomers demonstrate that increasing Na+ concentration up to approximately 0.2 M inhibits the formation of the Z conformation in the (m5dC-dG)n region of supercoiled plasmids. The results suggest that methylation may serve as a triggering mechanism for Z DNA formation in supercoiled DNAs.