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Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6–9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10–12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery. Structural and physiological studies show that the inner membrane protein PbgA is a crucial sensor of lipopolysaccharide (LPS) and regulates the activity of the LPS biosynthesis enzyme LpxC.

Background: We have discovered a new class of enzymes, that we name the nitrases, that catalyze the post-translation nitration of specific tyrosine residues of key proteins, several of which regulate important oncogenic pathways and are negatively prognostic in multiple cancer types. Materials And Methods: To identify protein substrates for 31 candidate nitrases, we incubated each nitrase with a 23,000 protein chip in a nitrase assay containing peroxynitrite and using anti-nitrotyrosine antibodies to detect the proteins that were nitrated. We then confirmed this nitration in separate biochemical assays and determined the specific site of nitration by LC-MS. Further, nitrases were evaluated in silico against TCGA gene expression and GISTIC copy number databases for specific cancer associations. Results: We have discovered that several proteins known to regulate tumor cell survival and/or evasion of immune surveillance are specifically nitrated by different nitrases and that their biochemical activities are regulated by this nitration. Pak4, a protein that has been implicated as an oncogenic driver, as well as in immune evasion is specifically nitrated by Nitrase #3 and increases its kinase activity. Interestingly, Nitrase #3 also nitrates Pak5, but not any of the other Pak family members, demonstrating high enzymatic specificity. Targeted inhibition or degradation of nitrated Pak4 or inhibition of Nitrase #3 may have therapeutic potential in oncology. Similarly, RhoA, a protein implicated in the oncogenic state of tumors is nitrated by Nitrase #12. This nitration activates RhoA biochemically and in cells. Bax is a specific substrate for Nitrase #11. Nitration of Bax reduces the cytochrome C release from the mitochondria induced by Bax. For each of these enzymes, we will develop specific inhibitors to determine their therapeutic potential in tumorigenesis. We further performed in silico analysis of nitrases for overall survival association in 17 cancer types. Utilizing TCGA RNA expression data and The Human Protein Atlas, nitrases were screened for association with overall survival in relation to nitrase RNA expression. Nitrases showing higher overall survival with low nitrase RNA expression and p. value Conclusions: A new class of enzymes we discovered offers multiple opportunities for development of effective chemotherapeutic agents against a variety of oncogenic targets and in specific cancers that show negative association of high nitrase expression. Citation Format: Arun Kashyap, Sami Hussein, Sahar Mazar, Kate Markham, Anthony Mastracci, Ritu Sharma, Sheerin K. Shahidi-Latham, Irene Griswold-Prenner. Enzymatic nitration in oncology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3941.

To better understand regional brain glycosphingolipid (GSL) accumulation in Gaucher disease (GD) and its relationship to neuropathology, a feasibility study using mass spectrometry and immunohistochemistry was conducted using brains derived from a GD mouse model (4L/PS/NA) homozygous for a mutant GCase (V394L [4L]) and expressing a prosaposin hypomorphic (PS-NA) transgene. Whole brains from GD and control animals were collected using one hemisphere for MALDI FTICR IMS analysis and the other for quantitation by LC-ESI-MS/MS. MALDI IMS detected several HexCers across the brains. Comparison with the brain hematoxylin and eosin (H&E) revealed differential signal distributions in the midbrain, brain stem, and CB of the GD brain versus the control. Quantitation of serial brain sections with LC-ESI-MS/MS supported the imaging results, finding the overall HexCer levels in the 4L/PS-NA brains to be four times higher than the control. LC-ESI-MS/MS also confirmed that the elevated hexosyl isomers were glucosylceramides rather than galactosylceramides. MALDI imaging also detected differential analyte distributions of lactosylceramide species and gangliosides in the 4L/PS-NA brain, which was validated by LC-ESI-MS/MS. Immunohistochemistry revealed regional inflammation, altered autophagy, and defective protein degradation correlating with regions of GSL accumulation, suggesting that specific GSLs may have distinct neuropathological effects.

The objectives of the present study were to characterize GNE-947 for its phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitory activities, in vitro anti-cell migration activity in Human Umbilical Vein Endothelial Cells (HUVEC), in vivo anti-neovascularization activity in laser induced rat choroidal neovascular (CNV) eyes, pharmacokinetics in rabbit plasma and eyes, and ocular distribution using Matrix-Assisted Laser Desorption Ionization Imaging mass spectrometry (MALDI-IMS) and autoradioluminography. Its PI3K and mTOR Ki were 0.0005 µM and 0.045 µM, respectively, and its HUVEC IC50 was 0.093µM. GNE-947 prevented neovascularization in the rat CNV model at 50 or 100 µg/eye with repeat dosing. Following a single intravenous injection at 2.5 and 500 μg/kg in rabbits, its plasma terminal half-lives (t1/2) were 9.11 and 9.59 hours, respectively. Following a single intravitreal injection of a solution at 2.5 μg/eye in rabbits, its apparent t1/2 values were 14.4, 16.3, and 23.2 hours in the plasma, vitreous humor, and aqueous humor, respectively. Following a single intravitreal injection of a suspension at 33.5, 100, 200 μg/eye in rabbits, the t1/2 were 29, 74, and 219 days in the plasma; and 46, 143, and 191 days in the eyes, respectively. MALDI-IMS and autoradioluminography images show that GNE-947 did not homogenously distribute in the vitreous humor and aggregated at the injection sites following injection of the suspension, which was responsible for the long t1/2 of the suspension due to slow dissolution process. This hypothesis was supported by pharmacokinetic modeling analyses. In conclusion, PI3K/mTOR inhibitor GNE-947 prevented neovascularization in a rat CNV model with t1/2 up to approximately six months following a single intravitreal injection of the suspension in rabbit eyes. SIGNIFICANCE STATEMENT GNE-947 is a potent PI3K/mTOR inhibitor and exhibits anti-CNV activity in rat eyes. The duration of GNE-947 in the rabbit eyes following intravitreal injection in a solution is short with t1/2 less than a day. However, the duration following intravitreal dose of a suspension is very long with t1/2 up to six months due to its low solubility and slow dissolution process. These results indicate that intravitreal injection of a suspension formulation for low solubility drugs can be used to achieve long-term drug exposure potentially useful for the treatment of wet AMD and other retinal diseases.

Aim: Objective of this study is to develop a robust multi-matrix LC–MS/MS for the quantitation of endogenous short-chain fatty acids (SCFA) biomarkers in human plasma and urine. Methods: Developed method utilizes stable isotope-labeled internal standards, high-throughput derivatization procedure for sample preparation and LC–MS/MS analysis using multiple reaction monitoring transitions in positive electrospray ionization mode. Results: Surrogate matrix method was used for quantitation. Accuracy, precision, parallelism, curve linearity, derivatization efficiency, stability and recovery were all evaluated, and the results were well within the acceptable criteria. Conclusion: SCFA levels in human plasma and urine of inflammatory bowel disease patients versus non-disease subjects were quantified and compared by LC–MS/MS.

The rate of enzyme degradation (kdeg) is an important input parameter for the prediction of clinical drug-drug interactions (DDIs) that result from mechanism-based inactivation or induction of cytochrome P450 (P450). Currently, a large range of reported estimates for CYP3A4 enzyme degradation exists, and consequently extensive uncertainty exists in steady-state predictions for DDIs. In the current investigations, the stable isotope labeled amino acids in culture technique was applied to a long-lived primary human hepatocyte culture, HepatoPac, to directly monitor the degradation of CYP3A4. This approach allowed selective isotope labeling of a population of de novo synthesized CYP3A4 and specific quantification of proteins with mass spectrometry to determine the CYP3A4 degradation within the hepatocytes. The kdeg estimate was 0.026 ± 0.005 hour-1 This value was reproduced by cultures derived across four individual donors. For these cultures, the data indicated that CYP3A4 mRNA and total protein expression (i.e., labeled and unlabeled P450s), and activity were stable over the period where degradation had been determined. This kdeg value for CYP3A4 was in good agreement with recently reported values that used alternate analytical approaches but also employed micropatterned primary human hepatocytes as the in vitro model.

Background: In early drug-discovery research, traditional techniques to analyze drug concentrations in tissues for bioanalytical needs include bead beaters and probe homogenization devices, but are not as effective for tough fibrous tissues. To prepare these tissues, the enzyme collagenase was used to digest the collagen fibers present in epithelial and connective tissue. Results: The benefits of tissue homogenization using a bead beater following collagenase treatment of samples, as opposed to using bead beating alone, was investigated. Matrix effect, recovery factor and stability with and without collagenase were assessed. Conclusion: Little to no effects on the quality and reliability of collagenase treated samples were observed. This enzymatic approach is a feasible and effective tool for tissue homogenization and subsequent analysis by LC-MS/MS.

Using structure-based design, a novel series of pyridone ERK1/2 inhibitors was developed. Optimization led to the identification of (S)-14k, a potent, selective, and orally bioavailable agent that inhibited tumor growth in mouse xenograft models. On the basis of its in vivo efficacy and preliminary safety profiles, (S)-14k was selected for further preclinical evaluation.

Preclinical and clinical evidence indicates that a subset of asthma is driven by type 2 cytokines such as interleukin-4 (IL-4), IL-5, IL-9, and IL-13. Additional evidence predicts pathogenic roles for IL-6 and type I and type II interferons. Because each of these cytokines depends on Janus kinase 1 (JAK1) for signal transduction, and because many of the asthma-related effects of these cytokines manifest in the lung, we hypothesized that lung-restricted JAK1 inhibition may confer therapeutic benefit. To test this idea, we synthesized iJak-381, an inhalable small molecule specifically designed for local JAK1 inhibition in the lung. In pharmacodynamic models, iJak-381 suppressed signal transducer and activator of transcription 6 activation by IL-13. Furthermore, iJak-381 suppressed ovalbumin-induced lung inflammation in both murine and guinea pig asthma models and improved allergen-induced airway hyperresponsiveness in mice. In a model driven by human allergens, iJak-381 had a more potent suppressive effect on neutrophil-driven inflammation compared to systemic corticosteroid administration. The inhibitor iJak-381 reduced lung pathology, without affecting systemic Jak1 activity in rodents. Our data show that local inhibition of Jak1 in the lung can suppress lung inflammation without systemic Jak inhibition in rodents, suggesting that this strategy might be effective for treating asthma.

Cobimetinib is a MEK inhibitor currently in clinical trials as an anticancer agent. The objectives of this study were to determine in vitro and in vivo if cobimetinib is a substrate of P-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp1) and to assess the implications of efflux on cobimetinib pharmacokinetics (PK), brain penetration, and target modulation. Cell lines transfected with P-gp or Bcrp1 established that cobimetinib was a substrate of P-gp but not a substrate of Bcrp1. In vivo, after intravenous and oral administration of cobimetinib to FVB (wild-type; WT), Mdr1a/b(-/-), Bcrp1 (-/-), and Mdr1a/b(-/-)/Bcrp(-/-) knockout (KO) mice, clearance was similar in WT (35.5 ± 16.7 mL/min/kg) and KO animals (22.0 ± 3.6 to 27.6 ± 5.2 mL/min/kg); oral exposure was also similar between WT and KO animals. After an oral 10 mg/kg dose of cobimetinib, the mean total brain to plasma ratio (Kp) at 6 h postdose was 0.3 and 0.2 in WT and Bcrp1(-/-) mice, respectively. In Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO mice and WT mice treated with elacridar (a P-gp and BCRP inhibitor), Kp increased to 11, 6, and 7, respectively. Increased brain exposure in Mdr1a/b(-/-) and Mdr1a/1b/Bcrp1(-/-) KO and elacridar treated mice was accompanied by up to ∼65% suppression of the target (pErk) in brain tissue, compared to WT mice. By MALDI imaging, the cobimetinib signal intensity was relatively high and was dispersed throughout the brain of Mdr1a/1b/Bcrp1(-/-) KO mice compared to low/undetectable signal intensity in WT mice. The efflux of cobimetinib by P-gp may have implications for the treatment of patients with brain tumors/metastases.

A multi-matrix hydrophilic interaction liquid chromatography tandem mass spectrometric method (HILIC-MS/MS) was developed for the quantitation of N-Acetyl Aspartic acid (NAA) using stable isotope labeled internal standard, D3-NAA in various biological matrices such as human plasma, human CSF, mouse plasma, brain and spinal cord. A high throughput 96-well plate format supported liquid extraction (SLE) procedure was developed and used for sample preparation. Mass spectrometric analysis of NAA was performed using selected reaction monitoring transitions in positive electrospray ionization mode. As NAA is endogenously present, a surrogate matrix approach was used for quantitation of NAA and the method was qualified over linear calibration curve range of 0.01-10μg/mL. Intra and inter assay precision indicated by percent relative standard deviation (%RSD) was less than 7.1% for low, medium, medium high and high QCs. The accuracy of the method ranged from 92.6-107.0% of nominal concentration for within-run and between-run for the same QCs. Extraction recovery of NAA and D3-NAA was greater than 76%. Stability of NAA was established in the above biological matrices under bench top (RT, 5h), freeze thaw (-20±10°C, 3 cycles) and moues/human plasma sample collection (Wet ice, RT) conditions. HILIC-MS/MS method was then used to quantify and compare the NAA levels in human plasma and CSF of ALS patients versus control human subjects. NAA CSF levels in control human subjects (73.3±31.0ng/mL,N=10) were found to be slightly higher than ALS patients (46.1±22.6ng/mL, N=10) (P=0.04). No differences were observed in NAA plasma levels in human control subjects (49.7±13.8ng/mL,N=9) as compared to ALS patients (49.6±8.1ng/mL, N=10) (P=0.983). NAA endogenous concentrations in mouse plasma, brain and spinal cord were found to be 243.8±56.8ng/mL (N=6), 1029.8±115.2μg/g tissue weight (N=5) and 487.6±178.4μg/g tissue weight (N=5) respectively.

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.

In drug discovery and development, in vitro absorption and metabolism assays along with in vivo pharmacokinetic (PK), pharmacodynamic (PD), and toxicokinetic (TK) studies are used to evaluate a potential drug candidate. More recently, imaging mass spectrometry approaches have been successfully reported to aid in the preclinical assessment of drug candidates, resulting in the rapid and noteworthy acceptance of the technique in pharmaceutical research. Traditionally, drug distribution studies via mass spectrometric imaging (MSI) are performed as targeted MS/MS analyses, where the analytes of interest, drug and/or metabolite, are known before the imaging experiment is performed. The study presented here describes a whole-body mass spectrometric imaging (WB-MSI) approach using a hybrid MALDI-LTQ-Orbitrap-MS to detect the distribution of reserpine at 2 h post a 20 mg/kg oral dose. This study effectively demonstrates the utility of obtaining accurate mass measurements across a wide mass range combined with postprocessing tools to efficiently identify drug and metabolite distributions without the need for any a priori knowledge.

Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has proven to be a quick, robust, and label-free tool to produce two-dimensional (2D) ion-density maps representing the distribution of a variety of analytes across a tissue section of interest. In addition, three-dimensional (3D) imaging mass spectrometry workflows have been developed that are capable of visualizing these same analytes throughout an entire volume of a tissue rather than a single cross-section. Until recently, the use of Fourier transform ion cyclotron resonance (FTICR) mass spectrometers for 3D volume reconstruction has been impractical due to software limitations, such as inadequate capacity to manipulate the extremely large data files produced during an imaging experiment. Fortunately with recent software and hardware advancements, 3D reconstruction from MALDI FTICR IMS datasets is now feasible. Here we describe the first proof of principle study for a 3D volume reconstruction of an entire mouse lung using data collected on a FTICR mass spectrometer. Each lung tissue section was analyzed with high mass resolution and mass accuracy, and considered as an independent dataset. Each subsequent lung section image, or lung dataset, was then co-registered to its adjacent section to reconstruct a 3D volume. Volumes representing various endogenous lipid species were constructed, including sphingolipids and phosphatidylcholines (PC), and species confirmation was performed with on-tissue collision induced dissociation (CID). Graphical Abstract ᅟ.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry has been adopted in the pharmaceutical industry as a useful tool to detect xenobiotic distribution within tissues. A unique sample preparation approach for MALDI imaging has been described here for the extraction and detection of cobimetinib and clozapine, which were previously undetectable in mouse and rat brain using a single matrix application step. Employing a combination of a buffer wash and a cyclohexane pre-extraction step prior to standard matrix application, the xenobiotics were successfully extracted and detected with an 8 to 20-fold gain in sensitivity. This alternative approach for sample preparation could serve as an advantageous option when encountering difficult to detect analytes.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM.

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.

DNA glycosylases catalyze scission of the N-glycosylic bond linking a damaged base to the DNA sugar phosphate backbone. Some of these enzymes carry out a concomitant abasic (apyrimidinic/apurinic (AP)) lyase reaction at a rate approximately equal to that of the glycosylase step. As a generalization of the mechanism described for T4 endonuclease V, a repair glycosylase/AP lyase that is specific for ultraviolet light-induced cis-syn pyrimidine dimers, a hypothesis concerning the mechanism of these repair glycosylases has been proposed. This hypothesis describes the initial action of all DNA glycosylases as a nucleophilic attack at the sugar C-1′ of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. It is proposed that the enzymes that are only glycosylases differ in the chemical nature of the attacking nucleophile from the glycosylase/AP lyases. Those DNA glycosylases, which carry out the AP lyase reaction at a rate approximately equal to the glycosylase step, are proposed to use an amino group as the nucleophile, resulting in an imino enzyme-DNA intermediate. The simple glycosylases, lacking the concomitant AP lyase activity, are proposed to use some nucleophile from the medium, e.g. an activated water molecule. This paper reports experimental tests of this hypothesis using five representative enzymes, and these data are consistent with this hypothesis.

Background: In early drug-discovery research, understanding the tissue distribution of drug at the site of action can help to predict the toxicity, efficacy and exposure level of the drug. The bottleneck of tissue analysis by LC–MS/MS is the time-consuming homogenization step. Results: Both mechanical and enzymatic techniques for mouse tissue homogenization were evaluated, which included bead beater, polytron and enzymatic digestion. Brain, bone marrow, kidney, spleen and liver tissues can be homogenized effectively using the bead beater alone. Lung and heart tissues were best treated with collagenase first and then homogenized by the bead beater. Conclusion: Homogenization conditions for seven mouse tissues have been evaluated and optimized. These findings will expedite the preparation of tissue samples for analysis.

The extracellular-signal-regulated kinases (ERK1 and ERK2) represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that commonly is activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling, such as receptor tyrosine kinase (RTK) activation. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Here, we present the discovery and characterization of GDC-0994, an orally bioavailable, small molecule inhibitor of ERK kinase activity. GDC-0994 is highly selective for ERK1 and ERK2, with biochemical potency of 1.1 nM and 0.3 nM, respectively. Daily, oral dosing of GDC-0994 results in significant single-agent activity in multiple in vivo cancer models, including KRAS-mutant and BRAF-mutant human xenograft tumors in mice. PD biomarker inhibition of phospho-p90RSK in these tumors correlates with potency in vitro and in vivo. In contrast to other published ERK inhibitors, GDC-0994 neither increases nor decreases phospho-ERK, suggesting that different ERK inhibitors have alternative mechanisms of action with respect to feedback signaling. Furthermore, we demonstrate a novel approach for targeting the oncogenic signaling through the RAS pathway by combining ERK and MEK inhibitors. GDC-0994 is currently in Phase I clinical development. Citation Format: Kirk Robarge, Jacob Schwarz, Jim Blake, Michael Burkard, Jocelyn Chan, Huifen Chen, Kang-Jye Chou, Dolores Diaz, John Gaudino, Stephen Gould, Jonas Grina, Xin Linghu, Lichuan Liu, Matthew Martinson, David A. Moreno, Christine Orr, Patricia Pacheco, Ann Qin, Kevin Rasor, Li Ren, Sheerin Shahidi-Latham, Jeffrey Stults, Francis Sullivan, Weiru Wang, Peter Yin, Aihe Zhou, Marcia Belvin, Mark Merchant, John G. Moffat. Discovery of GDC-0994, a potent and selective ERK1/2 inhibitor in early clinical development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT02-03. doi:10.1158/1538-7445.AM2014-DDT02-03

We report here that mouse embryos can exhibit a significant incidence of blastomere fragmentation at the two-cell stage. The incidence of this is influenced by both the maternal and paternal genotype. Embryos from C57BL/6 mothers exhibit a very low incidence of fragmentation at the two-cell stage in crosses involving males of C57BL/6, DBA/2, AKR/J, or SJL strains but exhibit a significantly increased incidence of fragmentation in crosses involving C3H/HeJ males. Increased fragmentation is seen in embryos from C3H/HeJ females crossed with C57BL/6 males but not with C3H/HeJ males. Embryos obtained from reciprocal (C57BL/6 x C3H/HeJ) F1 hybrid females also exhibit an increased incidence of fragmentation at the two-cell stage when the hybrid females are mated to either C57BL/6 or C3H/HeJ males. Interestingly, the results differ significantly between reciprocal F1 hybrid females, indicating a parental origin effect, possibly a result of either genomic imprinting or differences in mitochondrial origin. We conclude that the incidence of blastomere fragmentation at the two-cell stage in the mouse is under the control of more than one genetic locus. We also conclude that blastomere fragmentation is affected by both parental genotypes. These results are relevant to understanding the genetic control blastomere fragmentation, which may contribute to evolutionary processes, affect the success of procedures such as cloning, and affect the outcome of assisted reproduction techniques.

The time of onset of gene transcription in the mouse embryo is temporally regulated. A prominent feature of this regulation is a change during the one-cell stage from a transcriptionally nonpermissive state to a transcriptionally permissive state. During the early one-cell stage, the cytoplasm is either inadequate or suppressive for nuclear gene transcription, but by the late one-cell stage, the cytoplasm acquires the ability to support gene transcription either in endogenous nuclei or exogenous nuclei introduced microsurgically. We have investigated the role of protein synthesis in this cytoplasmic transition. Nuclei from two-cell stage embryos treated with alpha-amanitin were used to evaluate the transcriptional permissiveness of late one-cell stage cytoplasm, as indicated by the production of transcripts from four genes that are specifically transcribed at elevated rates during the two-cell stage. Two of these genes were transcribed following nuclear transfer to late one-cell stage cytoplasm, and two were not transcribed. Treatment of the recipient cytoplasm with cycloheximide to inhibit protein synthesis from the early to the late one-cell stage inhibited the transcription of the two genes that were transcribed in the untreated, late one-cell stage recipients. These results indicate that acquisition of the transcriptionally permissive state during the one-cell stage is facilitated by protein synthesis, and that the transcriptional permissiveness in the late one-cell stage cytoplasm is limited to certain genes.

Diploid androgenetic mouse embryos, possessing two sets of paternally inherited chromosomes, and control fertilized embryos were used to examine the relative effects of X chromosome number and parental chromosome origin on androgenone viability and X-linked gene expression. A significant difference in efficiency of blastocyst formation was observed between XX and XY androgenones in some experiments, but this difference was not uniformly observed. Significant effects of both X chromosome number and parental origin on X-linked gene expression were observed. Male and female control embryos expressed the XIST: RNA initially. This expression was followed by a preferential reduction in XIST: RNA abundance in male embryos, indicating that dosage compensation for the X chromosome may normally require the downregulation of XIST: RNA expression in male embryos, in conjunction with the production of stable XIST: transcripts in female embryos. By the late blastocyst stage, XX control embryos expressed significantly more XIST: RNA than did XY embryos. Unlike their normal counterparts, XX androgenones did not express significantly more XIST: RNA than did XY androgenones at the late blastocyst stage. Androgenones exhibited severe repression of the Pgk1 gene, but during development to the late blastocyst stage Pgk1 mRNA expression increased in XX androgenones and decreased in XY androgenones. Thus, the initial repression of the Pgk1 gene in XX androgenones was lost as the XIST: RNA declined in abundance, and this loss was correlated with a failure of XX androgenones to express significantly more XIST: RNA than did XY androgenones. These results indicate that androgenones may lack a factor that is expressed from the maternal genome and required for dosage compensation in preimplantation embryos. The results also indicate that early dosage compensation in preimplantation embryos may normally be reversible, thus providing flexibility to meet different developmental requirements of the embryonic and extraembryonic lineages.

Embryonic genome activation (EGA) in mice is sensitive to treatment with cycloheximide, indicating that protein synthesis plays an important role in mediating EGA. We hypothesized that regulated maternal mRNA recruitment may control the time of EGA by controlling the time of appearance of certain transcription factors (TFs). We also hypothesized that synthesis of other TFs may contribute to EGA independently of controlling the timing of EGA. To test these hypotheses, we used sucrose density gradient fractionation coupled to a quantitative reverse transcription-polymerase chain reaction method to compare polysomal mRNA abundances of specific TF mRNAs between metaphase II oocytes, 1-cell-stage embryos, and 2-cell-stage embryos. We observed a 2-cell-stage-specific increase in polysomal abundance of mouse TEA DNA binding domain 2 (mTEAD-2) mRNA, coincident with the first appearance of mTEAD activity in the early embryo. The mRNAs encoding Sp1, TATA binding protein, and cyclic AMP response element binding protein did not undergo translational recruitment, but exhibited differences in polysomal abundance. We also observed a continuous, high proportion in the polysomal fraction for the mRNA encoding ribosomal protein L23 mRNA, which contrasted with the patterns observed for other maternal transcripts. These observations are consistent with the hypothesis that regulated recruitment of maternal TF mRNAs may control the time of activation of some genes during EGA, and that continuous synthesis of other TFs, like Sp1, may facilitate EGA.

Activation of transcription within the embryonic genome (EGA) after fertilization is a complex process requiring a carefully coordinated series of nuclear and cytoplasmic events, which collectively ensure that the two parental genomes can be faithfully reprogrammed and restructured before transcription occurs. Available data indicate that inappropriate transcription of some genes during the period of nuclear reprogramming can have long-term detrimental effects on the embryo. Therefore, precise control over the time of EGA is essential for normal embryogenesis. In most mammals, genome activation occurs in a stepwise manner. In the mouse, for example, some transcription occurs during the second half of the one-cell stage, and then a much greater phase of genome activation occurs in two waves during the two-cell stage, with the second wave producing the largest onset of de novo gene expression. Changes in nuclear structure, chromatin structure, and cytoplasmic macromolecular content appear to regulate these periods of transcriptional activation. A model is presented in which a combination of cell cycle-dependent events and both translational and posttranslational regulatory mechanisms within the cytoplasm play key roles in mediating and regulating EGA.

Successful development of mammalian oocytes requires correct interactions between developing oocytes and associated granulosa cells. Development of oocyte-granulosa cell complexes from preantral follicles in vitro does not produce oocytes competent to develop to blastocysts at the same frequency as for oocytes that develop in vivo. Addition of either FSH or insulin to cultures of oocyte-granulosa cell complexes does not improve the frequency of blastocyst development, and the combination of both insulin and FSH is deleterious. Here, high-resolution 2-dimensional PAGE (2D-PAGE) and computerized gel image analysis were used to compare patterns of protein synthesis in cumulus cells and mural granulosa cells of small antral follicles, and then to assess effects of FSH and insulin on the differentiation of oocyte-associated granulosa cells (OAGCs) in vitro. Culture of OAGCs without FSH or insulin resulted in failure to synthesize many proteins at rates characteristic of cumulus cells. Either hormone used alone caused many cumulus cell proteins that were decreased in control cultures to be synthesized at nearly normal cumulus cell rates, and also caused the synthesis of other proteins to be increased or decreased. The two hormones added together produced the greatest change in protein synthetic pattern, including overexpression or underexpression of many proteins not affected by either hormone alone. Addition of these hormones to culture media thus appeared insufficient to elicit a normal cumulus cell phenotype in OAGCs and could lead to complex changes in protein synthesis that may be deleterious to oocyte development. The high-resolution 2D-PAGE approach described here should be a valuable tool in studies on oocyte and granulosa cell development in vitro, since phenotype can be evaluated globally through the display of over 1000 newly synthesized proteins rather than relying upon the expression of just a few genes.

Genomic imprinting in mammals results in the differential expression of maternal and paternal alleles of certain genes. Recent observations have revealed that the regulation of imprinted genes is only partially determined by epigenetic modifications imposed on the two parental genomes during gametogenesis. Additional modifications mediated by factors in the ooplasm, early embryo, or developing embryonic tissues appear to be involved in establishing monoallelic expression for a majority of imprinted genes. As a result, genomic imprinting effects may be manifested in a stage-specific or cell type-specific manner. The developmental aspects of imprinting are reviewed here, and the available molecular data that address the mechanism of allele silencing for three specific imprinted gene domains are considered within the context of explaining how the imprinted gene silencing may be controlled developmentally.

The newly fertilized preimplantation embryo depends entirely on maternal mRNAs and proteins deposited and stored in the oocyte prior to its ovulation. If the oocyte is not sufficiently equipped with maternally stored products, or if zygotic gene expression does not commence at the correct time, the embryo will die. One of the major abnormalities observed during early development is cellular fragmentation. We showed previously that cellular fragmentation in human embryos can be attributed to programmed cell death (PCD). Here, we demonstrate that the PCD that occurs during the 1-cell stage of mouse embryogenesis is likely to be regulated by many cell death genes either maternally inherited or transcribed from the embryonic genome. We have demonstrated for the first time the temporal expression patterns of nine cell death regulatory genes, and our preliminary experiments show that the expression of these genes is altered in embryos undergoing fragmentation. The expression of genes involved in cell death (MA-3, p53, Bad, and Bcl-xS) seems to be elevated, whereas the expression of genes involved in cell survival (Bcl-2) is reduced. We propose that PCD may occur by default in embryos that fail to execute essential developmental events during the first cell cycle.

Platelet aggregation and thrombus formation, resulting from disruption of a coronary artery plaque, play a critical role in the pathology of acute coronary syndromes. Currently, aspirin and heparin are administered to decrease platelet aggregation. The discovery of the platelet integrin receptor alpha(IIb)beta3, also known as the platelet glycoprotein (GP) IIb-IIIa receptor, is a breakthrough in antiplatelet therapy. The GP IIb-IIIa receptor is responsible for the crucial binding of fibrinogen to platelets, leading to cross-links between platelets and further platelet aggregation. Since the introduction of abciximab, the first GP IIb-IIIa-receptor antagonist, several other nonantibody agents have been studied for use in percutaneous transluminal coronary angioplasty and also in stent placement, treatment of unstable angina, and myocardial infarction.

HP1-like chromobox genes comprise an evolutionarily conserved family of genes that encode components of centromeric heterochromatin. In order to investigate the role of the murine HP1-like gene, M31, in heterochromatin formation we have isolated its gene and characterised its transcripts and protein products. PCR products that represent M31 transcripts were detected at the one-cell stage and were maternal in origin. Maternal provision of M31 transcripts may reflect a need for M31 in the formation of a functional centromere in order that there is proper segregation of chromosomes during the early cleavage divisions; studies in fission yeast and Drosophila have suggested a crucial role for HP1-like genes in centromere function. There are three protein products encoded by the M31 gene. Surprisingly, the two smaller products are found almost exclusively in the cytoplasm.

Previous studies indicated that members of the myc gene family may be essential for preimplantation development. Other studies revealed that preimplantation embryos lacking c-myc, N-myc, or L-myc are viable, indicating that these genes are either not essential for preimplantation development or can be substituted for functionally by other myc gene family members. To investigate the possible role of these genes during preimplantation development, we determined the temporal patterns of expression of four members of the myc gene family, genes encoding myc-associated proteins, and four putative MYC target genes. We observed a sequential pattern of myc gene expression, with the L-myc mRNA expressed as a maternal transcript, the c-myc mRNA expressed during the 4-cell through morula stages, and the B-myc mRNA expressed highly at the morula and blastocysts stages. B-myc was the predominant family member expressed during preimplantation development. The mxi mRNA was not detectable and the mad mRNA was detectable only as a maternal transcript. The max mRNA, however, was expressed both as a maternal mRNA and as an embryonic message throughout most of preimplantation development. Three putative MYC target genes (Odc, cyclin E, and prothymosin-alpha) were transcriptionally induced during the 2-cell stage, and their mRNAs increased sharply in abundance during development to the morula and blastocyst stages. Another putative MYC target gene, cyclin A, was expressed both as a maternal mRNA and as an embryonic transcript. These data support the view that the expression of myc target genes may be supported initially through the expression of maternally inherited MYC proteins and corresponding mRNAs and that subsequent stage-specific changes in expression of myc genes, myc-associated genes, and myc target genes may control early differentiative events around the time of implantation.

We recently reported the cloning and sequencing of the mouse A-myb proto-oncogene cDNA and the abundant expression of this mRNA primarily in the testis of adult mice. The A-myb mRNA is detectable by in situ hybridization specifically in the spermatogenic cells, and is downregulated during terminal differentiation. A low level of expression is observed in a few other tissues, including ovary, spleen and brain. We have extended those studies by examining A-myb and B-myb expression during testis development in the mouse. The A-myb and B-myb genes are both expressed in a cell- and stage-specific manner during testis development. The B-myb mRNA is expressed most highly in gonocytes of the fetal testis and in spermatogonia and early spermatocytes in the adult. B-myb expression decreases at day 18 post partum, coincident with the initial appearance of late pachytene spermatocytes. B-myb expression was also detectable in some interstitial cells of the fetal and adult testis. The A-myb mRNA was not detectable by in situ hybridization in fetal day 15.5 gonocytes but was detectable at a low abundance by RT-PCR in fetal and newborn mice. A-myb mRNA expression increased at post-natal day 10, when primary spermatocytes first appear. In the adult, the A-myb mRNA was expressed highly in a sub-population of spermatogonia and in primary spermatocytes, but was not detectable in spermatids. This expression of A-myb is consistent with the meiotic arrest that is observed in A-myb-deficient male mice. We conclude that B-myb may play a critical role in controlling the proliferation or differentiation of gonocytes and spermatogonia and possibly the somatic lineages as well, whereas A-myb is required for progression through the first meiotic prophase. These distinct roles for B-myb and A-myb during spermatogenesis may reflect distinct transactivation potentials of the two proteins. Further studies to determine the functions of A-myb and B-myb in the developing testis should improve our understanding of the molecular events associated with spermatogenesis and differentiation of the Sertoli and other somatic cell types of the testis.

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.

The design, chemical synthesis and biological activities of a nuclease-resistant, nontoxic bioactive 2-5A derivative, AA-etherA [i.e., adenylyl-(2'-5')-adenylyl-(2'-2")-9-[(2'-hydroxyethoxy)-methyl]adenine], are described as a new approach to the inhibition of breast cancer cell growth. AA-etherA inhibits DNA replication and cell division of both estrogen receptor positive (MCF-7) and estrogen receptor negative (BT-20) breast cancer cells in culture in a dose-dependent manner. Maximal inhibition in MCF-7 and BT-20 cells was obtained with 100 microM AA-etherA after four days of treatment, with an GI50 of 58 and 37 microM, respectively. AA-etherA is stable in the cytoplasm. Treated cells accumulate within the late G1/early S phase of the cell cycle and then progress only very slowly through S phase. AA-etherA does not activate RNase L, as do 2-5A and other 2-5A derivatives, nor does it increase p68 kinase (PKR) content of the cells. High resolution, two-dimensional protein gel electrophoresis reveals twofold or greater inhibition of synthesis of 92 proteins out of 682 proteins that were reproducibly detected as high quality spots with average rates of synthesis ofor = 20 p.p.m. in untreated cells. The specificity of the effects of AA-etherA on select proteins and its failure to activate RNase L indicate that AA-etherA does not act through a general effect on mRNA translation or stability, but rather inhibits cell proliferation through a block to DNA replication, with a concommitant reduction in the synthesis of specific proteins, some of which may be required for cell cycle transit. Two likely targets to account for the AA-etherA inhibition of DNA replication are DNA topoisomerase I, which is inhibited by AA-etherA in other cell lines, and thymidine kinase, which could be inhibited in a manner similar to the effect of acyclovir. These data indicate that 2-5A analogs, particularly bifunctional 2-5A analogs like AA-etherA, will be useful for controlling cancer cell growth. Further development of such 2-5A analogs may provide highly specific compounds for chemotherapy and chemoprevention.

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.

The interaction between endonuclease V, the cyclobutane pyrimidine dimer-specific N-glycosylase/abasic lyase from bacteriophage T4, and DNA was investigated by DNase I footprinting methods. The catalytically inactive mutant E23Q was found to interact with a smaller region of DNA at the abasic site analog, tetrahydrofuran, than at a thymine dimer site. Like the wild-type enzyme, the mutant contacted the DNA substrates primarily on the strand opposite the damage. The various complexes examined by footprinting techniques represent distinct points along the catalytic pathway of endonuclease V: before catalysis at a dimer, after N-glycosylase action but before abasic lyase action, and before catalysis at an abasic site. The differences between the footprints of the mutant and wild-type enzymes on both DNA substrates likely represent subtly different conformations within these complexes.

DNA glycosylases catalyze scission of the N-glycosylic bond linking a damaged base to the DNA sugar phosphate backbone. Some of these enzymes carry out a concomitant abasic (apyrimidinic/apurinic(AP)) lyase reaction at a rate approximately equal to that of the glycosylase step. As a generalization of the mechanism described for T4 endonuclease V, a repair glycosylase/AP lyase that is specific for ultraviolet light-induced cis-syn pyrimidine dimers, a hypothesis concerning the mechanism of these repair glycosylases has been proposed. This hypothesis describes the initial action of all DNA glycosylases as a nucleophilic attack at the sugar C-1' of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. It is proposed that the enzymes that are only glycosylases differ in the chemical nature of the attacking nucleophile from the glycosylase/AP lyases. Those DNA glycosylases, which carry out the AP lyase reaction at a rate approximately equal to the glycosylase step, are proposed to use an amino group as the nucleophile, resulting in an imino enzyme-DNA intermediate. The simple glycosylases, lacking the concomitant AP lyase activity, are propose to use some nucleophile from the medium, e.g. an activated water molecule. This paper reports experimental tests of this hypothesis using five representative enzymes, and these data are consistent with this hypothesis.

Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the α-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino-terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.

Genetic imprinting in mammals allows the recognition and differential expression of maternal and paternal alleles of certain genes. Recent results from a number of laboratories indicate that, at least for some genes, gametic imprints, which must exist in order to mark chromosomes or genes as having been transmitted via sperm or ovum, are not by themselves sufficient to determine allele expression. Other postfertilization events are required, and these events are subject to both tissue-specific and developmental stage-specific regulation. Changes in imprinted gene methylation during preimplantation and fetal life indicate that the establishment of additional allele-specific modifications is likely to contribute to imprinted regulation. Disruptions in imprinting processes, loss of imprints, and loss of nonimprinted alleles through uniparental disomy are likely to contribute to a variety of developmental abnormalities and pathological conditions in both mice and humans.

Bacteriophage T4 displays enhanced ultraviolet light (UV) resistance over other T-even bacteriophages. This enhanced resistance was initially ascribed to the v gene, now known as denV. The product of the denV gene, endonuclease V, has been shown to be responsible for initiating the removal of pyrimidine dimers from DNA. Initial characterization of purified endonuclease V revealed that the enzyme produces single-strand breaks in DNA at the site of UV-induced pyrimidine dimers, yet shows no activity towards unirradiated or heat-denatured DNA. The enzyme seems almost completely specific for cis-syn cyclobutane pyrimidine dimers, although recently it has been demonstrated to react with trans-syn dimers at approximately 1/100th the rate of the cis-syn dimer. DenV has been cloned and sequenced, allowing for expression of endonuclease V within Escherichia coli. The gene encodes a 138-amino acid protein with a predicted molecular mass of 16 kDa. Extensive biochemical and genetic studies have shown endonuclease V to possess four distinct activities: (1) a salt-dependent scanning or looping mechanism allowing for nontarget double-stranded DNA interactions; (2) a pyrimidine dimer-specific binding activity; (3) a pyrimidine dimer-specific DNA glycosylase activity; and (4) an apurinic/apyrimidinic (AP) lyase activity.

An exclusive correlation exists between the ectopic expression of the cell-surface marker, intestinal alkaline phosphatase (IAP), and the tumorigenic phenotype of segregants derived from suppressed, nontumorigenic HeLa x fibroblast cell hybrids. This specific association suggests that loss of tumor suppressor function is closely linked to the re-expression of IAP and, therefore, that IAP may be a critical oncogenic factor in these cells. To address this directly, we have used a HeLa IAP cDNA expression vector (pHIAP) to introduce constitutive IAP expression into a nontumorigenic HeLa x fibroblast cell hybrid. Sequence analysis of the HeLa IAP cDNA revealed 5 separate nucleotide alterations when compared to the native IAP cDNA sequence; however, none of these resulted in amino acid substitutions. Four pHIAP transfectants were analyzed for the presence of the intact integrated IAP cDNA and their relative expression levels of exogenous IAP mRNA and protein. The functional integrity of the cDNA-derived IAP product was confirmed by demonstrating proper enzymatic activity and localization to the extracellular membrane. The tumorigenic potentials of the pHIAP transfectants were assayed by s.c. injection into athymic nude mice. No tumors were observed, even after an 11-week incubation in animals. Therefore, expression of IAP in the nontumorigenic HeLa x fibroblast cell hybrid is not sufficient to confer the tumorigenic phenotype. Although ectopic IAP expression is unlikely to be functionally relevant to tumorigenicity in these hybrids, the significance of IAP as a tumor marker is still evident from its apparent strong association with a tumor suppressor locus.

Numerous studies have revealed stage specific alterations in protein synthesis that occur in mouse embryos. A thorough analysis of these changes has been hampered by limitations in the ability to resolve individual proteins, the ability to accurately quantify and manage data from two-dimensional gel images, and by variation in the staging of the embryos used. To learn more of the changes in protein synthesis that occur during early development, we constructed a protein database for the mouse embryo using the QUEST system of high-resolution, two-dimensional gel electrophoresis and computerized gel image analysis (Garrels, 1989, J. Biol. Chem., 264:526). Synchronous cohorts of embryos were labeled at 3 h intervals throughout the entire preimplantation period from fertilization to blastocyst stage in order to characterize in detail the changes in protein synthesis pattern that occur during normal preimplantation development. Additional samples were prepared from early post-implantation embryos, isolated inner cell mass and trophoblast cells, and cultured embryonic stem cells. These provide the means for identifying cell and tissue specific proteins and for characterizing changes in protein synthesis that accompany early cellular differentiation in the embryo. We present here a description of the mouse embryo database and discuss its potential usefulness to the study of mammalian embryogenesis.

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in genetically stable hybrids which are nontumorigenic and no longer express the HeLa tumor-associated antigen, intestinal alkaline phosphatase (IAP). Previous analysis of spontaneous segregants of the nontumorigenic hybrid have implicated the loss of one copy of human fibroblast chromosome 11 with reexpression of IAP and tumorigenicity. This observation suggests that a putative HeLa tumor suppressor gene(s) is located on chromosome 11 and that this gene may be a negative regulator of the IAP gene. We have isolated several gamma-ray-induced mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 that were specifically selected for reexpression of IAP to further investigate the potential linkage between IAP regulation and the putative tumor suppressor locus. The GIMs have a wide range of cell morphology and level of IAP expression (nearly a factor of 40). The tumorigenicity of the GIMs was examined by s.c. injection into nude mice and all were found to be tumorigenic. The tumor volume-doubling time is in the range of 4 to 8 days for all the cell lines; however, the lag time to reach 500 mm3 tumor volume was significantly longer when the GIM IAP activity was low (less than 20% relative activity), suggesting perhaps that there is a threshold level of IAP expression required for tumor formation and selection for high IAP expression in vivo. However, studies with tumor reconstitutes of the GIMs and transfection studies with an IAP complementary DNA expression vector indicate that high IAP expression alone is not sufficient to confer rapid tumor growth. Therefore, while the data lend strong support to the continued tight correlation between IAP reexpression and tumorigenicity and to our proposal that the tumor suppressor may negatively regulate the IAP gene, it suggests that selection for other gene activities may be responsible for aggressive tumor growth in this cell hybrid system.

Forty-one Diagnostic and Statistical Manual of Mental Disorders-III-Revised hypochondriacs were accrued from a primary care practice. Seventy-five control subjects were selected at random from among the remainder of the patients in the same clinic. All subjects completed a structured diagnostic interview and standardized self-report questionnaires. Medical morbidity was assessed with a medical record audit and with primary physicians' ratings. The hypochondriacal and comparison samples did not differ in aggregate medical morbidity, although the hypochondriacal sample had more undiagnosed complaints and nonspecific findings in their medical records. Within the comparison sample, higher levels of medical morbidity were associated with higher levels of hypochondriacal symptoms. This occurred primarily because the most serious medical disorders were associated with more bodily preoccupation, disease conviction, and somatization. Within the hypochondriacal sample, no correlation was found between the degree of hypochondriasis and the extent of medical morbidity.