Making the most of Papua New Guinea's biodiversity: Establishment of an integrated set of programs that link botanical survey with pharmacological assessment in "The Land of the Unexpected"

An integrated and coordinated set of programs has been established to meet ICBG goals in Papua New Guinea (PNG). Here we give an overview of the PNG ICBG and focus on the key elements and major steps taken to establish a program necessary for the pharmacological assessment of botanicals and traditional medicines in PNG and, by extrapolation, in other developing countries.

Correlation of biochemical effects and incorporation of 3-deazaguanine into nucleic acids to cytotoxicity in L1210 cells.

3-Deazaguanine, a tumor-inhibitory purine antimetabolite, is cytotoxic to L1210 leukemic cells in culture. The log percentage of viability correlated strongly (r2 = 0.986) with the product of the concentration of 3-deazaguanine, or [3-deazaguanine], and period of exposure (t) when [3-deazaguanine] was between 3 and 50 microM, and t was 12 or 24 h. We wished to relate this cytotoxicity to biochemical effects mediated by 3-deazaguanine. 3-Deazaguanine inhibited both DNA and protein synthesis, and both log DNA synthesis and log protein synthesis correlated inversely with [3-deazaguanine] X t and directly with cell viability (P less than 0.001). L1210 cells accumulated 3-deazaguanine 5'-triphosphate to a level of 1.5 nmol/10(6) cells. 3-Deazaguanine treatment had no effect on intracellular cytidine 5'-triphosphate levels, but reduced adenosine 5'-triphosphate and uridine 5'-triphosphate levels by 40% relative to control and guanosine 5'-triphosphate levels by 85% relative to control at a [3-deazaguanine] X t value at which 3-deazaguanine 5'-triphosphate accumulation was near maximal. Incorporation of 2-14C-labeled 3-deazaguanine into DNA and RNA, separated by Cs2SO4 density gradient centrifugation, was demonstrated. Incorporation into DNA was linear versus [3-deazaguanine] X t and correlated inversely with cell viability (P less than 0.001). These data suggest that 3-deazaguanine is anabolized and incorporated into DNA, and that this incorporation is related to decreased DNA synthesis and cell death. The decrease in protein synthesis and diminution of guanosine 5'-triphosphate levels following drug treatment may also contribute to the growth-inhibitory actions of 3-deazaguanine.

Cloning and characterization of a rat-specific repetitive DNA sequence.

A 2.1-kb EcoRI fragment of rat DNA has been cloned and sequenced. This fragment contained a repetitive element which was highly specific for rat DNA and widely dispersed throughout the rat genome. The repetitive element is homologous to a sequence found in the 3' end of the rat LINE family. Because of its high degree of species specificity and its heterodisperse distribution, this sequence provided a useful marker for rat DNA in DNA transfection experiments into mouse host cells.

Classical and nonclassical furo[2,3-d]pyrimidines as novel antifolates: synthesis and biological activities.

Classical antifolate analogues containing a novel furo[2,3-d]pyrimidine ring system which include N-[4-[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5- yl)methyl]amino]benzoyl]-L-glutamic acid (1) and its N-9 methyl analogue 2 were synthesized as potential dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Four nonclassical antifolates, 2,4-diamino-5-(anilinomethyl)furo[2,3-d]pyrimidines 3-6 with 3,4,5-trimethoxy, 3,4,5-trichloro, 3,4-dichloro, and 2,5-dimethoxy substituents, respectively, in the phenyl ring, were also synthesized as potential inhibitors of DHFRs including those from Pneumocystis carinii and Toxoplasma gondii, which are organisms responsible for opportunistic infections in AIDS patients. The classical and nonclassical analogues were obtained via nucleophilic displacements of the key intermediate 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with the appropriate (p-aminobenzoyl)-L-glutamate or substituted aniline. The key intermediate was in turn synthesized from 2,4-diamino-6-hydroxypyrimidine and 1,3-dichloroacetone. The final compounds were tested in vitro against rat liver, (recombinant) human, P. carinii, T. gondii, and Lactobacillus casei DHFRs. The classical analogues showed moderate to good DHFR inhibitory activity (IC50 10(-6)-10(-8) M) with the N-CH3 analogue 2 about twice as potent as 1. The nonclassical analogues were inactive with IC50S > 3 x 10(-5) M. The classical analogues were also evaluated as inhibitors of TS (L. casei, (recombinant) human and human CCRF-CEM), glycinamide ribonucleotide formyltransferase, and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and were found to be inactive against these enzymes. The classical analogues (particularly 2) were significantly cytotoxic toward a variety of tumor cell lines in culture. The nonclassical analogues were marginally active. Both classical compounds were good substrates for human folylpolyglutamate synthetase. Further evaluation of the cytotoxicity of 1 and 2 in CCRF-CEM cells and its sublines, having defined mechanisms of methotrexate (MTX) resistance, demonstrated that the analogues utilize the reduced folate/MTX-transport system and primarily inhibit DHFR and that poly-gamma-glutamylation was crucial to their mechanism of action. Protection studies in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin or the combination of thymidine plus hypoxanthine. Furthermore, for compounds 1 and 2, in contrast to MTX, the FaDu cells were better protected by thymidine alone than hypoxanthine alone, suggesting a predominantly antithymidylate effect.

Synthesis of 5-methyl-5-deaza nonclassical antifolates as inhibitors of dihydrofolate reductases and as potential antipneumocystis, antitoxoplasma, and antitumor agents.

A series of 2,4-diamino-5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines 4-9 were synthesized as 5-deaza nonclassical antifolates containing trimethoxy, dichloro-, or trichlorophenyl substitutions and a N-H, N-CH3, or N-CHO at the 10-position. The compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (P. carinii), Toxoplasma gondii (T. gondii), rat liver (RL), and Lactobacillus casei (L. casei); as inhibitors of T. gondii and P. carinii cell growth in culture; and as antitumor agents. The compounds were prepared by modifications of procedures for classical 5-deaza folates. 2,4-Diamino-5-methyl-6-[(3',4',5'-trimethoxy-N- methylanilino)methyl]pyrido[2,3-d]pyrimidine (5a) exhibited high potency as well as selectivity (compared to RL DHFR) for P. carinii and T. gondii DHFR. Compound 5a is one of the most potent and selective nonclassical folate inhibitors of T. gondii DHFR known. The N-10 formyl analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]pyrido-[2,3-d]pyrimidine (6a) had decreased potency, but it maintained high selectivity for T. gondii DHFR. The corresponding chloro-substituted analogues maintained potency or had decreased potency; N-10 substitution did not increase potency or selectivity to the extent observed in the 3',4',5'-trimethoxy series. Partial reduction of the B ring to afford the dihydro analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]-5,8-dihydropyrido[2,3-d]pyrimidine (7), its 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine analogue 8, and 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino)methyl]-5,6,7, 8- tetrahydropyrido[2,3-d]pyrimidine (9) resulted in a significant decrease in potency. In T. gondii cell culture inhibitory studies, 2,4-diamino-5-methyl-6-[(3',4',5'- trimethoxyanilino)methyl]pyrido[2,3-d]pyrimidine (4a), 5a, and 6a were less potent compared to their DHFR inhibitory potencies. Against P. carinii cells in culture, 4a and 5a at 10 micrograms/mL were as effective as the clinically used combination of trimethoprim/sulfamethoxazole (50/250 micrograms/mL). With the exception of the B ring reduced analogues 7-9, all of the compounds were significantly cytotoxic to leukemia CCRF-CEM cells in culture. The chloro-substituted analogues, in general, were more potent against a variety of other tumor cells in culture than the trimethoxy analogues. These results were corroborated by the preclinical tumor screening program at the National Cancer Institute where the most potent compound 2,4-diamino-5-methyl-6-[(3',4'-dichloroanilino)methyl]pyrido[2,3- d]pyrimidine (4b) was found to inhibit the growth of 26 tumor cell lines at an IG50 < 1.00 x 10(-8) M.

Inhibition of topoisomerase II catalytic activity by pyridoacridine alkaloids from a Cystodytes sp. ascidian: a mechanism for the apparent intercalator-induced inhibition of topoisomerase II.

Several new pyridoacridine alkaloids, dehydrokuanoniamine B (1), shermilamine C (2), and cystodytin J (3), in addition to the known compounds cystodytin A (4), kuanoniamine D (5), shermilamine B (6), and eilatin (7), were isolated from a Fijian Cystodytes sp. ascidian. Their structures were determined by analyses of spectroscopic data. These compounds along with a previously reported pyridoacridine, diplamine (8), showed dose-dependent inhibition of proliferation in human colon tumor (HCT) cells in vitro. All compounds inhibited the topoisomerase (TOPO) II-mediated decatenation of kinetoplast DNA (kDNA) in a dose-dependent manner. The pyridoacridines' ability to inhibit TOPO II-mediated decatenation of kDNA correlated with their cytotoxic potencies and their ability to intercalate into calf thymus DNA. These results suggest that disruption of the function of TOPO II, subsequent to intercalation, is a probable mechanism by which pyridoacridines inhibit the proliferation of HCT cells. Incorporation studies show that pyridoacridines disrupt DNA and RNA synthesis with little effect on protein synthesis. It appears that DNA is the primary cellular target of the pyridoacridine alkaloids. These results are consistent with those for known DNA intercalators.

Topoisomerase II-mediated DNA cleavage by adocia- and xestoquinones from the Philippine sponge Xestospongia sp.

Investigation of an orange Xestospongia sp. sponge collected at Cape Bolinao in northern Luzon, Philippines, yielded the known compounds adociaquinones A and B (1, 2) and six new metabolites, secoadociaquinones A and B (3, 4), 14-methoxyxestoquinone (5), 15-methoxyxestoquinone (6), 15-chloro-14-hydroxyxestoquinone (7), and 14-chloro-15-hydroxyxestoquinone (8). All compounds showed inhibition of topoisomerase II in catalytic DNA unwinding and/or decatenation assays. Furthermore, adociaquinone B showed activity in a KSDS assay, suggesting it inhibits the enzyme by freezing the enzyme-DNA cleavable complex. Interestingly, adociaquinone B did not displace ethidium bromide from DNA or unwind supercoiled DNA, implying it does not intercalate DNA.

Synthesis and pharmacological evaluation of isoindolo[1,2-b]quinazolinone and isoindolo[2,1-a]benzimidazole derivatives related to the antitumor agent batracylin.

The synthesis and pharmacological activity of isoindolo[1,2-b]quinazolin-12(10H)-ones and isoindolo[2,1-a]benzimidazoles related to batracylin are described. The acute toxicity of batracyclin has been associated with the formation of its N-acetyl metabolite which is a potent inducer of unscheduled DNA synthesis in rat hepatocytes. The desamino derivative and the 8-aza analog of batracylin retained the ability to inhibit topoisomerase II but did not induce unscheduled DNA synthesis. While less active than batracylin, these analogs were cytotoxic to CCRF CEM leukemia cells. The isoindolo[2,1-a]benzimidazole derivatives were inactive as topoisomerase II inhibitors and, in general, failed to exhibit comparable antitumor activity or to induce unscheduled DNA synthesis.

Evidence for the cloning of Deinococcus radiodurans DNA fragments that render Escherichia coli radiation resistant.

DNA from the radiation-resistant bacterium Deinococcus radiodurans was isolated and used to generate a cosmid library. This cosmid library was grown in Escherichia coli and radiation-resistant E. coli were isolated. Following exposure to 1000 Gy the radiation-resistant transformants exhibited a survival of approximately 10(-1) instead of the 10(-11) exhibited by the nontransformed E. coli. Smaller fragments of DNA were subcloned from the radiation-resistant E. coli; these fragments bestow similar levels of radiation resistance (ratio of slopes = 6.8) to native E. coli upon transfection.

Transfection of a human gene for the repair of X-ray- and EMS-induced DNA damage.

EM9 cells are a line of Chinese hamster ovary cells that are sensitive to killing by ethylmethanesulfonate (EMS) and X ray, since they are unable to repair the DNA damage inflicted by these agents. Through DNA-mediated gene transfer, human DNA and a selectable marker gene, pSV2neo, were transfected into EM9 cells. Resistant clones of transfected cells were selected for by growth in EMS and G418 (an antibiotic lethal to mammalian cells not containing the transfected neo gene). One primary clone (APEX1) and one secondary clone (TEMS2) were shown to contain both marker and human DNA sequences by Southern blot. In cell survival studies, APEX1 was shown to be as resistant to EMS and X ray as the parental cell type AA8 (CHO cells). TEMS2 cells were found to be partially resistant to EMS and X ray, displaying an intermediate phenotype more sensitive than AA8 cells but more resistant than EM9 cells. Alkaline elution was used to assess the DNA strand-break rejoining ability of these cells at 23 degrees C. APEX1 cells showed DNA repair capacity equal to that of AA8 cells; 75% of the strand breaks were repaired with a rejoining T 1/2 of 3 min. TEMS2 showed similar levels of repair but a T 1/2 for repair of 9 min. EM9 cells repaired only 25% of the breaks and showed a T 1/2 for repair of 16 min. The DNA repair data are consistent with the survival data in that the more resistant cell lines showed a greater capacity for DNA repair. The data support the conclusion that APEX1 and TEMS2 cells contain a human DNA repair gene.

Evidence against enhancement of the radioresistance of Escherichia coli by cloned Deinococcus radiodurans DNA.

Deinococcus radiodurans genomic DNA, introduced to Escherichia coli in cloning vectors, has been reported to produce radioresistant E. coli that can be selected by gamma irradiation. In this report prior results are reassessed experimentally, and additional studies are presented. Results to date suggest that the acquired radioresistance of E. coli selected by gamma irradiation does not stem from expression of stable plasmid-encoded D. radiodurans sequences, and that acquired radioresistance is not readily transmitted to naive (unirradiated) E. coli by transformation of plasmid recovered from the radioresistant isolates. Several interpretations are discussed.

Methylation of DNA guanine via the 1-carbon pool in dimethylnitrosamine-treated rats.

Treatment of rats with radioactive methionine and nonradioactive dimethylnitrosamine resulted in the formation of radioactive 7-methylguanine in rat-liver DNA. By comparing the specific activity of administered [14C-Me]-dimethylnitrosamine to the specific activity of isolated 7-methylguanine it was determined that following 20 mg/kg dimethylnitrosamine DNA methylation via the 1-carbon pool may account for up to 30% of the total 7-methylguanine formed.

Mutation, DNA labeling, and transformation of BHK-21/CL 13 cells by MNNG, and nitrosocimetidine.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been reported to induce BHK-21/Cl 13 cell growth in agar suspension. To determine if MNNG was also mutagenic to BHK cells, an ouabain-resistance mutation assay was established using these cells. In this system MNNG was compared to nitrosocimetidine (NC). MNNG and NC did induce ouabain-resistant mutations in BHK cells. The ability of the test compounds to methylate DNA in BHK cells was also determined, and both MNNG and NC yielded detectable levels of 7-methylguanine in treated cells. MNNG and NC were tested for the ability to transform BHK cells, and did. NC was found to be as effective a mutagen and transforming agent in BHK cells as MNNG when administered at equitoxic concentrations; approx. 4-fold less effective at equimolar concentrations.

DNA double-strand break repair in two radiation-sensitive mouse mammary carcinoma cell lines.

The capacity of two radiation-sensitive clones (SX9 and SX10) of the mouse mammary carcinoma cell line SR1 to rejoin radiation-induced DNA double-strand breaks (DSBs) was determined by pulsed-field agarose gel electrophoresis. DSBs were produced with equivalent efficiency in all three cell lines. Both the SX9 and SX10 cell lines demonstrated a significantly diminished capacity to rejoin radiation-induced DSBs. The fraction of the original DNA DSB damage remaining in the DNA of 20 Gy-exposed SR1, SX9 and SX10 cells after 6 h of 37 degrees C incubation was estimated to be 14, 82 and 54%, respectively. In addition the SX10 cell line exhibited enhanced cytotoxicity when exposed to the DNA topoisomerase II poison mitoxantrone. The results indicate that both the SX9 and SX10 cell lines are DNA DSB repair mutants.

The CHO XRCC1 mutant, EM9, deficient in DNA ligase III activity, exhibits hypersensitivity to camptothecin independent of DNA replication.

We have analyzed the X-ray-sensitive CHO mutant cell line EM9 for sensitivity to the topoisomerase I inhibitor comptothecin. These cells exhibit defective repair of single strand DNA breaks. Recently, EM9 were complemented the DNA ligase III interactive protein, XRCC1. Defective XRCC1 apparently accounts for the low DNA ligase III activity that may explain the single-strand break repair deficiency of EM9 cells. Here, we demonstrate cytotoxic hypersensitivity of EM9 cells following a brief camptothecin treatment. Both the S-phase and non-S-phase populations of EM9 exhibited camptothecin sensitivity relative to the parent cell line AA8. In AA8 cells, only the 55% of the population corresponding to the S-phase subpopulation were sensitive to camptothecin, while the remainder of the population were totally resistant to doses as high as 10 microM. The role of DNA replication in the camptothecin sensitivity was studied using the DNA polymerase inhibitor aphidicolin in co-treatment with camptothecin. Aphidicolin treatment fully protected AA8 cells from camptothecin cytotoxicity. In EM9 cells, aphidicolin protected the S-phase fraction to some degree but all the cells remained sensitive to camptothecin cytotoxicity. These results suggest that EM9 cells are sensitized to camptothecin by a mechanism that is independent of DNA replication and may be a consequence of the XRCC1 mutation or the associated deficiency in DNA ligase III activity. Mechanistic models for the replication-independent cytotoxicity of camptothecin in EM9 cells are discussed.

Nonenzymatic methylation of DNA by S-adenosylmethionine in vitro.

S-Adenosylmethionine was found to methylate DNA non-enzymatically to produce the same putative promutagenic and procarcinogenic lesions formed by carcinogenic chemical methylating agents. The formation of 7-methylguanine was confirmed by u.v. spectrophotometry, the formation of O6-methylguanine and 3-methyladenine was suggested by the cochromatography of radioactivity with standard bases. It is possible that this reaction may explain the presence of constitutive cellular enzymes specifically for the repair of methylated DNA, and may indicate the mechanism whereby methylated guanine is formed in the liver DNA of rats with chemically induced liver damage.

Heat sensitivity of HeLa S3 cell DNA topoisomerase II.

The sensitivity of HeLa DNA topoisomerase II to 45 degrees C heat shock was measured both in the intact cell and in vitro. In the intact cell, DNA topoisomerase II activity was estimated by measuring the formation and reversal of enzyme-DNA cleavable complexes by alkaline filter elution of cells exposed to the enzyme poison 4'-(9-acridinylamino)methanesulfon-m-anisidide). In vitro enzymatic activity was estimated by measuring changes in the topological state of plasmid and kinetoplast DNA produced by sonicates of nuclei from previously heated cells. The capacity of the enzyme to form, or reverse, enzyme-DNA cleavable complexes was inactivated during 45 degrees C heating with a reciprocal slope of 120 or 15 min, respectively. In vitro estimates of the activity of the enzyme from previously heated cells indicated that the enzyme was inactivated with a reciprocal slope of 99, 45, and 21 min after 45, 46 and 47 degrees C heating, respectively. DNA topoisomerase I activity was inactivated with a reciprocal slope of 130 min at 45 degrees C. The cumulative results indicate that during 45 degrees C heat shock, thermal inactivation of neither DNA topoisomerase I nor II is rate limiting for either cell survival or for DNA replication. While DNA topoisomerase II is resistant in situ to heat inactivation, in vivo assays indicate that the enzyme's capacity to function in the intact cell may be compromised by hyperthermic changes in the enzyme's environment.

Psammaplysin C: a new cytotoxic dibromotyrosine-derived metabolite from the marine sponge Druinella (= Psammaplysilla) purpurea.

The new, cytotoxic dibromotyrosine-derived metabolite psammaplysin C [3], in addition to the two known psammaplysins A [1] and B [2], was isolated from the marine sponge Druinella purpurea. All three compounds were found to possess moderate in vitro cytotoxicity towards the human colon tumor cell-line HCT116.

Transfectant CHO cells expressing O6-alkylguanine-DNA-alkyltransferase display increased resistance to DNA damage other than O6-guanine alkylation.

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea, Carmustine] is a nitrosourea that crosslinks DNA and is useful in cancer chemotherapy. Tumor cells resistant to BCNU produce high levels of O6-alkylguanine-DNA-alkyltransferase (AT), a protein that removes the O6-guanine adduct formed by BCNU prior to crosslinking. By the transfection of a human cosmid library into the Chinese hamster ovary cell line AA8, several transgenic cell lines which express the AT gene have been constructed. These 'BR' cells were isolated on the basis of their resistance to G-418 and BCNU. Like human mer+ strains, BR cells (relative to the parental AA8 cells) are approximately 500 times more resistant to the cytotoxic effects of 80 microM BCNU. Treatment with exogenous O6-methylguanine (O6MG), which depletes cellular AT, abolishes their BCNU resistance. Also consistent with the mer+ phenotype, BR cells are resistant to the mutagenic and killing activity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Treatment with exogenous O6MG, while reversing the resistance to MNNG mutation, does not reverse the resistance to MNNG killing. Unexpectedly, BR cells also exhibit resistance to killing by dimethylsulfate (DMS). The BR cells are not, however, detectably resistant to UV light. These results suggest that AT activity in mammalian cells is closely linked to the activity of other DNA repair pathways.

Cell-based screen for identification of inhibitors of tubulin polymerization.

This assay is based on morphological changes of rat glioma cells treated with db-cAMP. The db-cAMP treatment induces a tubulin-dependent change causing the cells to acquire a spherical shape. Pretreatment with tubulin inhibitors brings about the disintegration of tubulin polymer and/or prevents its polymerization. Cells with inhibited tubulin fail to respond to db-cAMP treatment. Cells treated with inhibitors of tubulin polymerization are then separated from the spherical cells by aspiration. A semiautomated scanning procedure evaluates the final culture density and yields graphical data.