One-pot Synthesis and Antitumor Activity of Unsymmetrical Terphenyls.

In this paper a simple and efficient method for the unsymmetrical terphenyls via sequential one-pot Suzuki coupling reactions using Pd(OAc)2 without isolation of the intermediate is described. The prepared terphenyls were found to possess potent anticancer properties against a panel of cancer cells which includes A549, HeLa, MCF7, DU145, HT29 and BxPC-3. Structural similarity with combretastatin A4, these terphenyls disrupted the tubulin polymerization in vitro and destabilized the microtubules in cells. Flow cytometry studies indicated growth arrest of cells in the G2/M phase of the cell cycle corresponding to antimitotic action. Furthermore, compound 4c showed potent anti-mitotic activity even in zebrafish model and could likely be a potential therapeutic compound as it is active both in in vitro and in vivo.

Protein phosphorylation is a regulatory mechanism for O6-alkylguanine-DNA alkyltransferase in human brain tumor cells.

The biochemical regulation of human O6-alkylguanine-DNA alkyltransferase (AGT), which determines the susceptibility of normal tissues to methylating carcinogens and resistance of tumor cells to many alkylating agents, is poorly understood. We investigated the regulation of AGT by protein phosphorylation in a human medulloblastoma cell line. Incubation of cell extracts with [gamma-32P]ATP resulted in Mg(2+)-dependent phosphorylation of the endogenous AGT. Immunoprecipitation after exposure of the cells to 32P-labeled inorganic phosphate showed that AGT exists as a phosphoprotein under physiological conditions. Western analysis and chemical stability studies showed the AGT protein to be phosphorylated at tyrosine, threonine, and serine residues. Purified protein kinase A (PKA), casein kinase II (CK II), and protein kinase C (PKC) phosphorylated the recombinant AGT protein with a stoichiometry of 0.15, 0.28, and 0.44 (mol phosphate incorporated/mol protein), respectively. Residual phosphorylation of the endogenous AGT by the PKs present in cell homogenates and phosphorylation of the recombinant AGT by purified serine/threonine kinases, PKA, PKC, and CK II reduced AGT activity by 30-65%. Conversely, dephosphorylation of cell extracts by alkaline phosphatases stimulated AGT activity. We also identified consensus phosphorylation motifs for many cellular kinases, including PKA and CK II in the AGT protein. These data provide the first and conclusive evidence of AGT phosphorylation and suggest that reversible phosphorylation may control the activity of this therapeutically important DNA repair protein in human normal and cancer cells.

Deletions and rearrangements inactivate the p16INK4 gene in human glioma cells.

Structural alterations in the p16INK4 gene were examined in early passage human glioma cell lines and related to the expression of p16 transcripts and protein. Using the Southern blot approach, we observed both homozygous and hemizygous deletions, as well as rearrangements of the p16 and p15 genes in 5 of the 7 cell lines (71%). Two cell lines, MGR3 and HBT28, revealed hemizygous deletion of the p16 and p15 genes combined with indistinguishable rearrangements of the remaining p15-p16 locus that resulted in loss of exon 2 sequences for p15 and p16, but retention of p16 exon 1; neither of these cell lines expressed p16 mRNA. Data for a third cell line, MGR2, indicated a similar, but unique rearrangement involving the p15 and p16 genes. MGR2, which retained a single wild-type p15-p16 locus, showed expression of p16 transcript, but not of p16 protein as indicated by Western blot analysis. All the glioma cell lines expressed similar levels of the retinoblastoma protein and no amplification of the cyclin-dependent kinase 4 gene. These results demonstrate that human glioma cells contain p16 gene microdeletions and rearrangements that contribute to inactivation of the cell cycle regulatory protein.

Nuclear magnetic resonance studies of polyamine binding to a defined DNA sequence.

The binding of spermine to the self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been studied by nuclear magnetic resonance spectroscopy. Free spermine gives narrow resonance lines and positive nuclear overhauser effects are observed between the spermine protons, as expected for a small molecule rotating freely in solution. In the spermine-DNA complex, there was no broadening of the spermine spectrum and very weak positive nuclear overhauser effects were observed, indicating that the spermine still has a remarkably short rotational correlation time. Spermine induced no changes in the DNA spectrum beyond those found upon addition of other salts. Although spermine interacts with DNA with a binding constant of approximately 10(6) at the low ionic strength under which these experiments were performed, it appears that the nature of the complex and the lifetime of the ligand on the DNA are such that the mobility of the spermine molecule is effectively independent of that of the DNA molecule.

Enforced expression of wild-type p53 curtails the transcription of the O(6)-methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents.

We used isogenic human tumor cell lines to investigate the specific and direct effects of wild-type (wt) p53 on the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that confers tumor resistance to many anticancer alkylating agents. A p53-null, MGMT-proficient lung tumor cell line (H1299) was engineered to express wt p53 in a tetracycline-regulated system. High levels of p53 induction achieved by tetracycline withdrawal were accompanied by G(1) cell cycle arrest without significant apoptosis in this cell line. p53 accumulation resulted in a gradual and dramatic loss of MGMT mRNA, protein, and enzyme activity, whose levels were undetectable by day 3 of induction. The loss of MGMT protein was, however, not due to its degradation because the ubiquitin-promoted in vitro degradation of MGMT, which mediates the cellular disposal of the repair protein, was not altered by p53. Run-on transcription assays revealed a significant reduction in the rate of MGMT gene transcription. The negative regulation of MGMT expression by wt p53 was confirmed in two other human isogenic cell lines, namely, the GM47.23 glioblastoma, which contains a dexamethasone-inducible wt p53, and the H460 lung cancer cell line, in which wt p53 had been inactivated by the human papillomavirus E6 protein. Furthermore, a panel of four human tumor cell lines, including gliomas with wt p53 status, displayed markedly lower levels of MGMT gene transcripts than those having p53 mutations. Induction of wt p53 in these models led to a 3- and 2-fold increase in sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide, respectively, which generate the MGMT-repairable O(6)-alkyl adducts in DNA. These results demonstrate that p53 is a negative regulator of MGMT gene expression and can create a MGMT-depleted state in human tumors similar to that achieved by O(6)-benzylguanine, a potent inhibitor of MGMT currently undergoing clinical trials. Thus, our study exposes an additional benefit associated with p53 gene therapy and provides a strong biochemical rationale for combining the MGMT-directed alkylators with p53 gene transfer to achieve improved antitumor efficacy.

Activity and distribution of the cysteine prodrug activating enzyme, 5-oxo-L-prolinase, in human normal and tumor tissues.

5-Oxo-L-prolinase (OPase), a key enzyme of the gamma-glutamyl cycle, has the ability to metabolize L-2-oxothiazolidine-4-carboxylic acid (OTC) to cysteine, and thereby increase intracellular glutathione (GSH) levels. This strategy of GSH elevation can be potentially exploited to reduce normal tissue toxicity of anticancer agents, provided that sufficient differences exist in OPase levels between normal and malignant tissues. In this study, therefore, we quantitated OPase activity in primary specimens of matched and unmatched human normal and tumor (lung, breast, kidney, colon and ovary) tissues using a newly developed non-radioactive OPase assay, based on the production of cysteine from OTC. The rank order of OPase activity in extracts of 24 normal tissues examined was kidney > lung, breast and colon > ovary. OPase activity was present in all 37 tumor samples, but at variable levels. Tumor OPase levels were generally equivalent to those in their normal tissue counterparts, with the notable exception of Wilms' tumors, which had markedly lower levels than normal kidney (P < 0.02). However, when 14 matched tumor and adjacent normal tissues were compared, OPase levels were significantly higher in normal specimens than tumors for individual patients (P < 0.005). These higher normal tissue/tumor OPase ratios suggest that OTC may be useful in decreasing normal tissue toxicity, at least, for some tissues during cancer therapy.

Formation and disappearance of DNA interstrand cross-links in human colon tumor cell lines with different levels of resistance to chlorozotocin.

Three human colon tumor (HCT) cell lines, designated C, Moser and 116, exhibiting a gradation of resistance to chlorozotocin, a glucose-linked chloroethylnitrosourea (1-, 2.9-, and 5.8-fold respectively) were examined to assess the determinants of drug sensitivity. Although the O6-alkylguanine-DNA transferase content was relatively higher in the most resistant 116 cells than in the sensitive cell line C, its level in Moser cells did not correlate with the intermediate chlorozotocin sensitivity. Glutathione content in these tumor cell lines did not show a parallelism with drug resistance. The ethidium bromide fluorescence assay was used to quantitate the kinetics of DNA interstrand cross-link formation and its removal after drug exposure. The peak levels of DNA interstrand cross-links induced in HCT cells correlated with their resistance to chlorozotocin with cross-link indices of 0.03, 0.10 and 0.20, respectively, for 116, Moser and C cell lines. All three cell lines demonstrated DNA cross-link repair to different extents. While the smaller number of cross-links formed in resistant 116 and Moser cells were eliminated in a rapid phase of repair, the lesions formed at a much greater frequency in C cells remained largely unrepaired. These results draw attention to the role of increased DNA cross-link repair as a mechanism of nitrosourea resistance in the HCT cells studied.

Stimulation and inhibition of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced strand breaks and interstrand cross-linking in Col E1 plasmid deoxyribonucleic acid by polyamines and inorganic cations.

The influence of various polyamines and metallic cations on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced DNA single-strand breaks and DNA interstrand cross-linking was in Col E1 plasmid using electrophoretic techniques. Spermidine and spermine (0.4 to 1.5 mM concentration range) markedly stimulated BCNU-induced DNA nicking, whereas putrescine had no effect on the nicking process. In contrast to the polyamines, BCNU-induced DNA nicking was decreased by the three inorganic cations, Na+ (100 and 200 mM), Mg2+ (0.5 and 1.5 mM), and Co3+ (NH3)6 (0.2 and 0.4 mM), with the trivalent hexamminecobalt ions being most inhibitory. When the monofunctional N-methyl-N-nitrosourea (MNU) was used (instead of the bifunctionally active BCNU) to alkylate Col E1 DNA, nicking of the DNA was inhibited by spermidine. Furthermore, the ability of chloroethylated Col E1 DNA to form interstrand cross-links after treatment with BCU was inhibited by 0.5 mM spermidine and 0.5 mM spermine, both concentrations within the intracellular range. Putrescine at 3-6 mM only marginally stimulated DNA cross-linking. In comparison, the inorganic cations all enhanced Col E1 DNA cross-linking by BCNU, with the rank order of cross-link stimulation being Mg2+, Na+, and Co3+ (NH3)6. These results provide evidence that polyamines can interact with DNA to modulate chloroethylnitrosourea-induced DNA damage and that the interaction is not only a function of the charge on the polyamine molecule but also of the chemical structure of the polyamine.

Coordinate up-regulation of cyclin-dependent kinase 4 and its inhibitor p16(INK4) in human glioma cells following chloroethylnitrosourea-induced DNA damage.

Anomalies in the genes of the cell cycle regulators, p16(INK4) and CDK4 are highly frequent in human gliomas and other cancers, however, the extent to which these defects are involved in regulating the response of tumor cells to DNA damaging agents is not clear. In this study, using three human malignant glioma cell lines, MGR1, MGR3, and U87MG, we examined changes in gene expression of p16 and/or of its specific target CDK4 following damage of the cellular genome by the chemotherapeutic bifunctional alkylating agent, 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Exposure of the cells to 50 mu M BCNU for 24 h induced a significant level of DNA interstrand cross-links in all cell lines. In MCR1 cells (p16(+), Rb+), over the 24 h period, a steady increase in p16 (mRNA and protein) and CDK4 (protein) was observed. The increase in CDK4 and p16 proteins occurred in parallel, and that the two proteins accumulated in complex with each other, resulting in marked inhibition of CDK4 kinase activity. In MGR3 and U87MG cells, both of which lack functional p16 protein (p16(-), Rb+), BCNU, however increased the CDK4 protein levels. In all three cell lines, despite the differences in p16 gene status, BCNU exposure caused significant blockade of the cells at G(2)/M phase of the cell cycle. The coordinated enhancement of the target (CDK4) and the inhibitor (p16) in cellular response to genomic injury may reflect an attempt of the cells to continue progression through the cell cycle (via CDK4), while triggering the cell cycle arrest (via p16), required for the orderly repair of the damage to the genome.

Effects of ethanol ingestion on glucose transporter-1 protein and mRNA levels in rat brain.

In the normal adult brain, glucose provides 90% of the energy requirement, as well as substrate for nucleic acid and lipid synthesis. We have previously observed that ethanol impairs hexose uptake by rat astrocytes in culture. In the present study, male Sprague-Dawley rats, 200-250 g, were fed liquid diet in which 36% of the calories were derived from ethanol (EF) for 4 weeks. Controls were fed ad libitum (AF) or pair-fed (PF) an equicaloric diet without ethanol. Blood glucose levels did not differ between the groups at the time of study. Glucose transport by brain plasma membranes was characterized by cytochalasin B binding and showed a slight increase in transporter number (mean +/- SEM of 4 experiments = 76.4 +/- 2.5 pmoles/mg protein in EF vs. 69.5 +/- 1.0 in PF) with no change in affinity (1.8 +/- 0.1 nM-1 in EF and 1.6 +/- 0.1 in PF). Glucose transporter, GLUT-1, was increased on Western blots. In contrast, Northern analysis of cortical tissue, using a rat brain glucose transporter cDNA insert (1.59 kb Bgl II fragment of pSPGT-1), showed a 23 to 35% decrease in steady-state levels of glucose transporter mRNA. GLUT-1 mRNA, localized in brain sections by in situ hybridization histochemistry, showed marked reductions in choroid plexus and hippocampus following ethanol treatment. Ethanol appears to have multiple effects on brain GLUT-1.

Decreased glucose transporter 1 gene expression and glucose uptake in fetal brain exposed to ethanol.

Using pregnant rats fed equicaloric liquid diets (AF, and libitum-fed controls; PF, pair-fed controls; EF, ethanol-fed), we have previously shown that maternal alcoholism produces a specific and significant decrease of glucose in the fetal brain, which is accompanied by growth retardation. To further define the mechanisms of ethanol-induced perturbations in fetal fuel supply, we have examined (i) the uptake of 2-deoxyglucose (2-DG) by dissociated brain cells from fetal rats that were exposed to ethanol in utero and (ii) the steady-state levels of the glucose transporter-1 (GT-1) mRNA. A 9% decrease in brain weight (P less than 0.001) and a 54.8% reduction in 2-DG uptake into brain cells (P less than 0.02) were found in offspring of EF mothers compared to the AF group. Brain weight correlated with the rate of 2-DG uptake (P less than 0.05). Northern blot analysis showed a 50% reduction of GT-1 mRNA in EF brain relative to that in the AF and PF groups. We conclude that glucose transport into the brain is an important parameter altered by maternal ethanol ingestion.

DNA interstrand crosslinking and strand break repair in human glioma cell lines of varying [1,3-bis(2-chloroethyl)-1-nitrosourea] resistance.

The production of DNA interstrand crosslinks (ISC) by BCNU and other bifunctional alkylators and the effects of these drugs on the repair of radiation-induced DNA-single strand breaks (SSB) were studied in two human glioblastoma used to assess both DNA-ISCs and DNA-SSBs. BCNU-treated UWR2 and UWR3 cells showed a significant BCNU dose-dependent increase in radiation-induced DNA-SSBs at 6 hrs post-drug treatment, and at 100 microM BCNU DNA-ISC was completely masked in UWR2 cells. There was no enhancement of radiation-induced DNA-SSBs in both cell lines after treatment with cis-DDP, CHZ, or MNU. In the capillary clonogenic cell assay, UWR2 cells were 3.2 times more resistant than UWR3 cells; 0(6)-methylguanine-DNA methyltransferase activity was also 1.8 times higher in UWR2 than in UWR3. Our data suggest caution in the use of the standard alkaline elution technique (with 6 hrs between drug exposure and irradiation) to measure BCNU-induced DNA-ISC induction in highly BCNU-resistant cell lines. We provide evidence that the synergism between BCNU and radiation in the generation of DNA-SSBs is the result of low DNA-SSB repair capacity of the cells, and is further potentiated by the carbamoylating action of BCNU.

Targeting MDM2-p53 interaction for cancer therapy: are we there yet?

Inactivation of the tumor suppressor p53 and/or overexpression of the oncogene MDM2 frequently occur in human cancers, and are associated with poor prognosis, advanced forms of the disease, and chemoresistance. MDM2, the major negative regulator of p53, induces p53 degradation and inactivates its tumor suppressing activity. In turn, p53 regulates MDM2 expression. This MDM2-p53 negative feedback loop has been widely studied and presents an attractive target for cancer therapy, with a few of the inhibitors of this interaction already having advanced into clinical trials. Additionally, there is an increasing interest in understanding MDM2's p53-independent activities in carcinogenesis and cancer progression, which may also have implications for cancer therapy. This review aims to highlight the various roles that the MDM2-p53 interaction plays in cancer, the p53 independent oncogenic activities of MDM2 and the various strategies that may be used to target MDM2 and the MDM2-p53 interaction. We will summarize the major preclinical and clinical evidences of MDM2 inhibitors for human cancer treatment and make suggestions to further improve efficacy and safety of this interesting class of cancer therapeutics.

Modulation of the relaxing activity of Escherichia coli topoisomerase I by single-stranded DNA binding proteins.

Removal of negative superhelical turns in ColE1 plasmid DNA by Escherichia coli topoisomerase I was markedly enhanced by the presence of single-stranded DNA binding protein from E. coli. A lack of species specificity makes unlikely the possibility of physical association between topoisomerase I and single-stranded DNA binding proteins. Stabilization of single-stranded regions in supercoiled DNA by single-stranded DNA binding protein would appear to be the basis of the enhancement of topoisomerase activity.

Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions.

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.

Enzymic synthesis of sym-homospermidine in Lathyrus sativus (grass pea) seedlings.

An enzyme catalysing the synthesis of sym-homospermidine from putrescine and NAD+ with concomitant liberation of NH3 was purified 100-fold from Lathyrus sativus (grass pea) seedlings by affinity chromatography on Blue Sepharose. This thiol enzyme had an apparent mol.wt. of 75000 and exhibited Michelis-Menten kinetics with Km 3.0mM for putrescine. The same enzyme activity could also be demonstrated in the crude extracts of sandal (Santalum album) leaves, but with a specific activity 15-fold greater than that in L. sativus seedlings.

Enzymic conversion of agmatine to putrescine in Lathyrus sativus seedlings. Purification and properties of a multifunctional enzyme (putrescine synthase).

The participation of a multifunctional enzyme (a single polypeptide with multiple catalytic activities (14)) has been demonstrated in the conversion of agmatine to putrescine in Lathyrus sativus seedlings. This enzyme (putrescine synthase) with inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase, and carbamate kinase has been purified to homogeneity and has Mr = 55,000. In the presence of inorganic phosphate, the enzyme catalyzed the stoichiometric conversion of agmatine and ornithine to putrescine and citrulline, respectively. The different activities associated with the enzyme copurified with near constancy in their specific activity. The enzyme catalyzed phosphorolysis and arsenolysis of N-carbamyl putrescine. The multifunctionality of putrescine synthase was also supported by 1) activity staining, 2) intact transfer of the ureido-14C group from labeled NJ-carbamyl putrescine to ornithine to form citrulline, and 3) the affinity of the enzyme toward structurally and functionally related affinity matrices. An agmatine cycle is proposed wherein N-carbamyl putrescine arising from the agmatine iminohydrolase reaction is converted to putrescine and citrulline, with the ureido group of N-carbamyl putrescine being transferred intact to ornithine. Preliminary results indicate that this series of reactions is also present in other plants.

Alterations in the activities of DNA topoisomerase enzymes and O6-methylguanine-DNA-methyltransferase in septic rat liver.

To understand the genomic changes contributing to the various metabolic derangements in sepsis and septic shock, we measured the activities of the following liver enzymes intimately associated with DNA function: (1) DNA topoisomerases I and II (topo I and topo II) controlling DNA conformation in mammalian nuclei, and (2) O6-methylguanine-DNA-methyltransferase (MT) capable of removing the methyl groups from the O6-position of guanine in DNA. We found that in septic rat livers the specific activities (units/mg protein) of topo II and MT were elevated by 1.4- and 1.6-fold, respectively, over the sham-operated controls (P less than 0.001). There was no significant difference in topo I activity. We believe that peritonitis sepsis alters topo II levels modulating the selective pretranscriptional changes in chromatin and that MT functions as a cellular stress protein.

Differential modulation by spermidine of reactions catalyzed by type 1 prokaryotic and eukaryotic topoisomerases.

In the absence of DNA aggregation, spermidine inhibited the relaxation of negatively supercoiled DNA by Escherichia coli topoisomerase I at concentrations of the polyamine normally found intracellularly. Spermidine also curtailed the cleavage of negatively supercoiled ColE1 DNA by the enzyme in the absence of Mg2+. On the contrary, knotting of M13 single-stranded DNA circles catalyzed by topoisomerase I was stimulated by the polyamine. Relaxation of supercoiled DNA by eukaryotic type 1 topoisomerases, such as calf thymus topoisomerase I and wheat germ topoisomerase, was significantly stimulated by spermidine in the same range of concentrations that inhibited the prokaryotic enzyme. In reactions catalyzed by S1 nuclease, the polyamine enhanced the digestion of single-stranded DNA and inhibited the nicking of negatively supercoiled DNA. These results suggest that spermidine modifies the supercoiled duplex substrate in these reactions by modulating the degree of single strandedness.