Effects of gossypol on DNA replication in mammalian cells

Gossypol, a binaphthalene compound, possesses male infertility effects. However, its mechanism of action and effects on somatic cells are not yet understood. The purpose of this study was to examine the effects of gossypol on mammalian cell growth and DNA replication, using tissue culture cells (HeLa) as an in vivo model.^ Gossypol inhibited DNA synthesis in HeLa cells at low doses, without affecting RNA or protein synthesis. This caused cells to accumulate in S phase without affecting cells in other phases of the cell cycle. The inhibition of DNA synthesis was both dose- and time-dependent. This irreversible block was associated with a decrease in HeLa plating efficiency. Gossypol did bind to DNA but did not measurably affect its ability to serve as a template for DNA polymerase $\alpha$, the major replicative enzyme. Only in the absence of serum could gossypol induce single-strand DNA breaks in HeLa cells; no DNA-DNA or DNA-protein crosslinks were formed.^ Gossypol exhibited dose-dependent inhibition of a number of eukaryotic and prokaryotic replicative DNA polymerases both in vitro and in vivo. This inhibition was kinetically non-competitive with respect to the DNA template and dNTP substrates. Both a filter binding assay and polyacrylamide gel electrophoresis were used to study gossypol binding to DNA polymerase. Inhibition resulted from drug binding to two adjacent amino acid residues on the enzyme. Binding was found to be irreversible and mediated through either non-covalent interactions or by Schiff's base formation between the aldehyde groups of gossypol and the $\varepsilon$-NH$\sb2$ groups of amino acid residues on the polymerase. Structure-function studies using eleven gossypol derivatives revealed that both aldehyde and hydroxyl groups function independently to effect inhibition of DNA polymerase and DNA replication. The activities of DNA polymerase $\beta$ and ribonucleotide reductase were also inhibited by increasing gossypol concentrations.^ These studies demonstrate that the gossypol-mediated inhibition of DNA replication is due in part to inhibition of key replicative enzymes, such as DNA polymerase $\alpha$. The study of DNA polymerase may serve as a model for the interaction of enzymes with gossypol, a drug which may prove useful as a chemotherapeutic agent. ^

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

REPSA: A method for determining the sequence specificity of DNA binding ligands

We developed a novel combinatorial method termed restriction endonuclease protection selection and amplification (REPSA) to identify consensus binding sites of DNA-binding ligands. REPSA uses a unique enzymatic selection based on the inhibition of cleavage by a type IIS restriction endonuclease, an enzyme that cleaves DNA at a site distal from its recognition sequence. Sequences bound by a ligand are protected from cleavage while unprotected sequences are cleaved. This enzymatic selection occurs in solution under mild conditions and is dependant only on the DNA-binding ability of the ligand. Thus, REPSA is useful for a broad range of ligands including all classes of DNA-binding ligands, weakly binding ligands, mixed populations of ligands, and unknown ligands. Here I describe REPSA and the application of this method to select the consensus DNA-binding sequences of three representative DNA-binding ligands; a nucleic acid (triplex-forming single-stranded DNA), a protein (the TATA-binding protein), and a small molecule (Distamycin A). These studies generated new information regarding the specificity of these ligands in addition to establishing their DNA-binding sequences. ^

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

RNA binding characteristics and overall topology of the vaccinia virus polyadenylation complex

mRNA 3′ polyadenylation is central to mRNA biogenesis in prokaryotes and eukaryotes, and is implicated in numerous aspects of mRNA metabolism, including efficiency of mRNA export from the nucleus, message stability, and initiation of translation. However, due to the great complexity of the eukaryotic polyadenylation apparatus, the mechanisms of RNA 3 ′ end processing have remained elusive. Although the RNA processing reactions leading to polyadenylated messenger RNA have been studied in many systems, and much progress has been made, a complete understanding of the biochemistry of the poly(A) polymerase enzyme is still lacking. My research uses Vaccinia virus as a model system to gain a better understanding of this complicated polyadenylation process, which consist of RNA binding, catalysis and polymerase translocation. ^ Vaccinia virus replicates in the cytoplasm of its host cell, so it must employ its own poly(A) polymerase (PAP), a heterodimer of two virus encoded proteins, VP55 and VP39. VP55 is the catalytic subunit, adding 30 adenylates to a non-polyadenylated RNA in a rapid processive manner before abruptly changing to a slow, non-processive mode of adenylate addition and dissociating from the RNA. VP39 is the stimulatory subunit. It has no polyadenylation catalytic activity by itself, but when associated with VP55 it facilitates the semi-processive synthesis of tails several hundred adenylates in length. ^ Oligonucleotide selection and competition studies have shown that the heterodimer binds a minimal motif of (rU)2 (N)25 U, the “heterodimer binding motif”, within an oligonucleotide, and its primer selection for polyadenylation is base-type specific. ^ Crosslinking studies using photosensitive uridylate analogs show that within a VP55-VP39-primer ternary complex, VP55 comes into contact with all three required uridylates, while VP39 only contacts the downstream uridylate. Further studies, using a backbone-anchored photosensitive crosslinker show that both PAP subunits are in close proximity to the downstream −10 to −21 region of 50mer model primers containing the heterodimer binding motif. This equal crosslinking to both subunits suggests that the dimerization of VP55 and VP39 creates either a cleft or a channel between the two subunits through which this region of RNA passes. ^ Peptide mapping studies of VP39 covalently crosslinked to the oligonucleotide have identified residue R107 as the amino acid in close proximity to the −10 uridylate. This helps us project a conceptual model onto the known physical surface of this subunit. In the absence of any tertiary structural data for VP55, we have used a series of oligonucleotide selection assays, as well as crosslinking, nucleotide transfer assays, and gel shift assays to gain insight into the requirements for binding, polyadenylation and translocation. Collectively, these data allow us to put together a comprehensive model of the structure and function of the polyadenylation ternary complex consisting of VP39, VP55 and RNA. ^

Analysis of the E1-ubiquitin activating enzyme, Uba1, in cell death and tissue growth in Drosophila

Ubiquitination is an essential process involved in basic biological processes such as the cell cycle and cell death. Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Subsequently, ubiquitin is transferred to target proteins via ubiquitin ligases (E3). Defects in ubiquitin conjugation have been implicated in several forms of malignancy, the pathogenesis of several genetic diseases, immune surveillance/viral pathogenesis, and the pathology of muscle wasting. However, the consequences of partial or complete loss of ubiquitin conjugation in multi-cellular organisms are not well understood. Here, we report the characterization of nba1, the sole E1 in Drosophila. We have determined that weak and strong nba1 alleluias behave genetically different and sometimes in opposing phenotypes. For example, weak uba1 alleluias protect cells from cell death whereas cells containing strong loss-of-function alleluias are highly apoptotic. These opposing phenotypes are due to differing sensitivities of cell death pathway components to ubiquitination level alterations. In addition, strong uba1 alleluias induce cell cycle arrest due to defects in the protein degradation of Cyclins. Surprisingly, clones of strong uba1 mutant alleluias stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner resulting in severe overgrowth phenotypes in the mosaic fly. I have determined that the observed overgrowth phenotypes were due to a failure to downregulate the Notch signaling pathway in nba1 mutant cells. Aberrant Notch signaling results in the secretion of a local cytokine and activation of JAK/STAT pathway in neighboring cells. In addition, we elucidated a model describing the regulation of the caspase Dronc in surviving cells. Binding of Dronc by its inhibitor Diap1 is necessary but not sufficient to inhibit Dronc function. Ubiquitin conjugation and Uba1 function is necessary for the negative regulation of Dronc. ^

Modulation of cytochrome P450 enzyme activity and PAH -induced skin carcinogenesis by naturally occurring coumarins

The present study was designed to determine the potential anticarcinogenic activity of naturally occurring coumarins and their mechanism of action. The results indicated that several naturally occurring coumarins including bergamottin, coriandrin, imperatorin, isopimpinellin, and ostruthin, to which humans are routinely exposed in the diet, were effective inhibitors and/or inactivators of CYP1A1-mediated ethoxyresorufin-O-dealkylase (EROD) or CYP2B1-mediated pentoxyresorufin-O-dealkylase (PROD) in mouse liver microsomes. In addition, bergamottin and corandrin were also found to be inhibitors of purified human P450 1A1 in vitro. Further studies with coriandrin revealed that this compound was a mechanism-based inactivator of P450 1A1 and covalently bound to the P450 1A1 apoprotein. In cultured mouse keratinocytes, bergamottin and coriandrin effectively inhibited the B(a) P metabolism and significantly decreased covalent binding of B(a) P and DMBA to keratinocyte DNA and anti-diol-epoxide-DNA adducts derived from both B(a) P and DMBA in keratinocytes. The data from in vivo experiments showed that bergamottin and coriandrin were potent inhibitors of covalent binding of B (a) P to epidermal DNA and the formation of (+) anti BPDE-DNA adduct, whereas imperatorin and isopimpinellin were more potent inhibitors of covalent binding of DMBA to epidermal DNA. The ability of coumarins to inhibit covalent binding of B (a) P to DNA in mouse epidermis was positively correlated with their inhibitory effect P450 1A1 in vitro, while the inhibitory effect of coumarins on covalent binding of DMBA to epidermal DNA was positively correlated with their inhibitory effects on P450 2B1 and negatively to their inhibitory activity toward P450 1A1. The data from tumor experiments indicated that bergamottin, ostruthin, and coriandrin inhibited tumor initiation by B (a) P in a two-stage carcinogenesis protocol. Bergamottin was most effective in this regard and produced a dose dependent inhibition of papilloma formation in these experiments. In addition, imperatorin was an effective inhibitor of skin tumorigenesis induced by DMBA in SENCAR mouse skin using both a two-stage and a complete carcinogenesis protocol. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B (a) P. The results to date demonstrate that several naturally occurring coumarins possess the ability to block tumor initiation and tumorigenesis by PAHs such as B (a) P and DMBA through inhibition of the P450s involved in the metabolic activation of these hydrocarbons. A working model for the involvement of specific P450s in the metabolic activation of these two PAHs was proposed. ^

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^

Role of the glutathione S -transferase P1 (GSTP1) electrophile binding site (H-site) in protection from doxorubicin -mediated cytotoxicity

The hypothesis addressed in this project was that novel variants of naturally occurring human glutathione S-transferase P1 (GSTP1) can be created by random mutagenesis of the GSTP1 active site to yield polypeptides with increased enzymatic activity against electrophilic substrates. Specifically, the mutant proteins would metabolize and inactivate selected electrophiles more efficiently than wild-type GSTP1 and confer significant cytoprotection, as measured by reduced apoptosis and increased clonogenic survival. Glutathione S-transferase P1, a major electrophile metabolizing and detoxifying enzyme, is encoded by a polymorphic genetic locus. This locus contains nucleotide transitions in the region encoding the active site of the peptide that yields proteins with significant structural and functional differences. The method of Degenerate Oligonucleotide Mediated Random Mutagenesis (DOMRM) was used to generate cDNAs encoding unique GSTP1 polypeptides with mutations within electrophile binding site (H-site) while leaving the glutathione binding site unaffected. A prokaryotic expression library of the mutant GSTP1 polypeptides was created and screened for increased resistance to cisplatin. This screen resulted in the isolation of 96 clones representing 22 distinct mutant cDNA sequences. To investigate the effects of the changes in the H-site on the biological activity of GSTP1, the cDNA of wild-type GSTP1c and two of the identified mutants were stably transfected into human LNCaP-Pro5 prostate cancer cells that do not endogenously express GSTP1. Wild-type transfectants were resistant to doxorubicin-induced apoptosis and displayed increased clonogenic survival compared to vector controls. However, contrary to the hypothesis, in both assays the mutant transfectants were no more resistant to doxorubicin than the wild-type transfectants. To elucidate the mechanisms underlying GSTP1-mediated survival, an in-vitro assay was developed to determine whether active GSTP1 protein directly metabolizes doxorubicin by conjugation to reduced glutathione (GSH). Although GSH did promote the appearance of a unique doxorubicin conjugate, conjugate formation was not substantially increased by the addition of GSTP1 in a variety of reaction conditions. ^