RNAi-based screening of the human kinome identifies Akt-cooperating kinases: a new approach to designing efficacious multitargeted kinase inhibitors.

Tumors comprise genetically heterogeneous cell populations, whose growth and survival depend on multiple signaling pathways. This has spurred the development of multitargeted therapies, including small molecules that can inhibit multiple kinases. A major challenge in designing such molecules is to determine which kinases to inhibit in each cancer to maximize efficacy and therapeutic index. We describe an approach to this problem implementing RNA interference technology. In order to identify Akt-cooperating kinases, we screened a library of kinase-directed small interfering RNAs (siRNAs) for enhanced cancer cell killing in the presence of Akt inhibitor A-443654. siRNAs targeting casein kinase I gamma 3 (CSNK1G3) or the inositol polyphosphate multikinase (IPMK) significantly enhanced A-443654-mediated cell killing, and caused decreases in Akt Ser-473 and ribosomal protein S6 phosphorylation. Small molecules targeting CSNK1G3 and/or IPMK in addition to Akt may thus exhibit increased efficacy and have the potential for improved therapeutic index.

Complex gel permeation assays for screening combinatorial libraries.

Gel permeation methods have been commonly used to screen combinatorial libraries synthesized on a solid support. We report here three screens of combinatorial libraries using gel permeation assays. These include a simple enzymatic assay to identify inhibitors of the influenza enzyme neuraminidase, and two more complex assays designed to screen for inhibitors of the interleukin-8 (IL-8)-IL-8 receptor and the urokinase-urokinase receptor interactions, respectively. The IL-8 ligand-receptor assay makes use of IL-8 receptor-expressing cells attached to a membrane, thus enabling washing steps as part of the assay. The urokinase ligand-receptor assay employs an enzyme-linked immunosorbent assay-type format, previously thought to be amenable only to well-based assays. The results of these three screens are reported here, including the discovery of a novel series of acyclic inhibitors of neuraminidase. The development of complex assays in a gel permeation format allows for the routine screening of combinatorially as well as noncombinatorially made compound collections against virtually any kind of target, and is being widely used in our high throughput screening operations.

Covalent bonding of bay-region diol epoxides to nucleic acids.

Although the solution chemistry of diol epoxides is now fairly well understood, a great deal remains to be elucidated regarding their reaction in the presence of DNA. Not only DNA but also small molecules are capable of sequestering diol epoxides in aqueous solutions with equilibrium constants on the order of 10(2)-10(4) M-1. In the case of DNA, at least two major families of complexes are presently recognized, possibly the result of groove binding vs. intercalation. As is the case for diol epoxides free in solution, the complexed diol epoxides undergo solvolysis to tetraols and in some cases possibly to keto diols as well. Fractionation between covalent bonding and solvolysis from within the complex(s) is determined more by the nature of the parent hydrocarbon from which the diol epoxide is derived than any other factor. Studies of a wide variety of alkylating and arylating agents have show that practically every potentially nucleophilic site on DNA can serve as a target for modification. In the case of the diol epoxides, practically all of the modification occurs at the exocyclic amino groups of the purine bases. In contrast to the diol epoxides, other epoxides such as those derived from aflatoxin B1, vinyl chloride, propylene, 9-vinylanthracene, and styrene preferentially bind to the aromatic ring nitrogens N-7 in guanine and N-3 in adenine (cf. Chadha et al., 1989). Molecular modeling as well as the spectroscopic evidence suggests that the hydrocarbon portion of the diol epoxides lies in the minor groove of DNA when bound to the exocyclic 2-amino group of guanine and in the major groove when bound to the exocyclic 6-amino group of adenine. Detailed conformational analysis of adducted DNA should prove to be extremely valuable in developing mechanistic models for the enzymatic processing of chemically altered DNA. At present, the critical lesion or lesions responsible for induction of neoplasia remains obscured by the large number of apparently noncritical adducts which form when polycyclic hydrocarbon diol epoxides bond to DNA.

Effects of route of administration on tissue distribution of DNA adducts in mice: comparison of 7H-dibenzo(c,g)carbazole, benzo(a)pyrene, and 2-acetylaminofluorene.

The environmental pollutant 7H-dibenzo(c,g)carbazole (DBC) has been shown to be a potent carcinogen in various mouse tissues, but displays an unusual degree of hepatocarcinogenicity. We have previously reported that in accord with this activity, mouse liver is the target organ for DBC-DNA binding, with total levels being up to 2700 times greater than in extrahepatic tissues after s.c. administration. To elaborate on this finding, we have directly compared the tissue distribution of DNA damage by three diverse aromatic carcinogens, DBC, benzo(a)pyrene (BP), and 2-acetylaminofluorene (AAF). Following a single topical, p.o., or s.c. administration of 80 mumol/kg of test compound to male BALB/c mice, a 32P-postlabeling assay showed the total number of DBC adducts in liver DNA to be 11-138 times that in kidney, lung, or skin DNA. The degree of hepatic adduction varied as a function of the route of administration, with the highest occurring after topical application and the lowest after s.c. injection. The tissue preference for AAF and BP adducts varied with the route of administration and was much less than for DBC adducts, except that topical application of BP gave DNA adduct levels in skin that were 91-218 times greater than in other tissues. For a given tissue and route of administration, DNA adduction by DBC was 1.7- to 950-fold greater than that by BP and AAF, except in skin where the level of DNA adducts from BP was 3 to 4 times that from DBC. We conclude that (a) DBC exhibits an exceptional and unique preference for liver DNA adduction after different routes of administration; (b) DBC is more potent overall than BP or AAF in causing tissue DNA damage; and (c) for each of the three carcinogens, the route of exposure is a much less important factor than the nature of the carcinogen in determining the tissue distribution of covalent DNA damage.

Gastric evacuation of the lemon shark Negaprion brevirostris (Poey) under controlled conditions.

Gastric evacuation of the juvenile lemon shark, Negaprion brevirostris, a tropical inshore apex predator, was studied in the laboratory under conditions of 25 degrees C and 32% salinity. Sharks were starved for 48-96 h after which they were fed a 2.7% body weight meal of fish fillet. Gastric evacuation was assessed by stomach lavage every 3 h for 24 h thereafter. Results showed that the gut is emptied in about 24 h with fillets being removed from the stomach following the equation Y = Yoe-bt, where Y is percent of meal recovered at time t, Yo is the initial meal size, and b is the instantaneous rate of evacuation. The exponential function of gastric evacuation in the lemon shark is similar to that in teleosts, but the kinetics are slower. The caloric density of the meal remaining in the stomach increased, approaching that of protein in the first 12 h after feeding, as carbohydrates (glycogen) were digested. After 12 h the caloric value dropped as protein was digested, suggesting an orderly and efficient intake of food energy by the lemon shark.

Comparison of 32P-postlabeling and high pressure liquid chromatographic analyses for 7,12-dimethylbenz[a]anthracene--DNA adducts.

[3H]7,12-Dimethylbenz[a]anthracene-modified DNA obtained from mouse cells in culture was enzymatically hydrolyzed to nucleoside 3'-phosphates, postlabeled with [32P]phosphate, and the carcinogen-modified nucleoside bisphosphates were separated by thin layer chromatography. Each adduct spot was eluted, dephosphorylated and the resulting [3H]nucleoside adducts were analyzed by high pressure liquid chromatography so that the structural information available for the liquid chromatographic peaks could be applied to the spots obtained from the postlabeling procedure. After this cross referencing, specific dihydrodiol epoxide-nucleotide adducts can now be monitored by the postlabeling technique.

N-methylation reduces the DNA-binding activity of 7H-dibenzo[c,g]carbazole approximately 300-fold in mouse liver but only approximately 2-fold in skin: possible correlation with carcinogenic activity.

N-methyl-dibenzo[c,g]carbazole (MeDBC) lacks the potent hepatocarcinogenic activity in mice characteristic for 7H-dibenzo[c,g]carbazole (DBC), while both compounds are local carcinogens, leading to papilloma and carcinoma formation in skin after topical application. Because DNA binding is considered an essential step in the initiation of chemical carcinogenesis, the DNA adduction by MeDBC was compared with that by DBC in mouse liver and skin via a 32P-postlabeling technique. Both compounds elicited chromatographically similar adducts in liver; however, the extent of total DNA binding of DBC was 343- and 265-fold greater than that of MeDBC 24 h after topical and i.p. administration, respectively, of a 37 mumol/kg dose. In skin, the adduct pattern elicited by either compound after topical application was different from that seen in liver, and three of four adducts derived from MeDBC were chromatographically distinct from those produced by DBC. Quantitative analysis revealed that total adduction in skin by DBC was 2.3-fold higher than by MeDBC. When the adduct levels were compared between liver and skin, topically applied MeDBC bound preferentially to skin versus liver DNA by a factor of 10, while the opposite was true for DBC. These data are in agreement with the carcinogenicity reported for DBC and MeDBC and support the hypothesis that the extent of covalent DNA modification by these compounds is associated with their biological activity. We conclude that an unsubstituted nitrogen is essential for the genotoxic activity of DBC in liver but not skin. The results also demonstrate the potential of the 32P-postlabeling assay in predicting the organotropism of closely related carcinogens.

32P-postlabeling analysis of DNA adduction in mice by synthetic metabolites of the environmental carcinogen, 7H-dibenzo[c,g]carbazole: chromatographic evidence for 3-hydroxy-7H-dibenzo[c,g]carbazole being a proximate genotoxicant in liver but not skin.

The DNA adduction by the environmental carcinogen 7H-dibenzo[c,g]carbazole (DBC) and chemically synthesized 2-OH, 3-OH, and 4-OH metabolites of DBC was investigated in liver and skin of female CD-1 mice. After topical application to the skin of 37 mumol/kg of DBC or the phenolic metabolites, DNA adducts were measured by a 32P-post-labeling assay employing carrier-free [gamma-32P]ATP and ATP-deficient conditions. In liver, DBC produced four major and several minor chromatographically distinct adducts of as yet undetermined chemical structure. The adduct pattern elicited by 3-OH-DBC was qualitatively similar to the DBC adduct pattern, while this was not the case for 2-OH-DBC and 4-OH-DBC. On the basis of co-chromatography experiments under various conditions, the DBC and 3-OH-DBC adducts appeared identical, and the total of adduction elicited by these compounds in liver was substantial. Similar results were observed when DBC or 3-OH-DBC were administered i.p. As a major difference between the two compounds, one 3-OH-DBC adduct (no. 3) was 4.4- and 7.0-fold lower than the corresponding DBC adduct after i.p. and topical dosing, respectively. In skin, DBC produced two major adduct fractions after topical application, one of which could be chromatographically resolved into three subcomponents. Prominent adducts produced in skin DNA by each of the three metabolites were different from those elicited by DBC, and the level of adduction by the metabolites was significantly lower than that by DBC. Comparison of the skin and liver DBC-DNA adduct patterns after topical application of DBC showed that only one of the four major chromatographically resolved skin adducts corresponded to a major liver adduct (no. 3), and that total adduction in liver was 13.5-fold higher than in skin. These results suggested that activation of DBC to DNA-binding compounds in liver occurs through at least two pathways with 3-OH-DBC being a proximate carcinogen involved in the formation of most of the adducts; 3-OH-DBC and the other two phenolic metabolites investigated play a minor role, if any, in the formation of DBC-DNA adducts in skin; metabolic activation of DBC to DNA-binding compounds in liver and skin appears to follow pathways that are different in terms of both the chemical nature and the amount of the adducts formed; and DBC and 3-OH-DBC exhibit a strong preference for liver versus skin DNA.

Human cell-mediated cytotoxicity, mutagenicity, and DNA adduct formation of 7H-dibenzo(c,g)carbazole and its N-methyl derivative in diploid human fibroblasts.

The aromatic N-heterocyclic 7H-dibenzo(c,g)carbazole (DBC), like polynuclear aromatic hydrocarbons, is a potent inducer of local sarcomas, papillomas, and respiratory tumors, but unlike such compounds it also induces hepatomas. The N-methyl derivative of DBC, N-methyl-dibenzo(c,g)carbazole (MeDBC), also induces sarcomas, papillomas, and respiratory tumors but at a lower frequency than DBC. However, MeDBC lacks hepatocarcinogenic potential, suggesting that DBC undergoes metabolic activation at its carbon atoms and also at the nitrogen position and that the N-7 position plays a role in liver carcinogenesis by DBC. We compared the cytotoxic and mutagenic potential of DBC and MeDBC using a human epithelial cell-mediated activation assay. Repair-deficient, diploid human fibroblasts derived from a xeroderma pigmentosum (XP) patient were used as target cells, and a human hepatoma cell line, Hs703T, was used as a source of exogenous metabolism. Resistance to 6-thioguanine served as the genetic marker for mutations. The Hs703T cells, but not the target XP cells, activated DBC and MeDBC into forms capable of interacting with DNA. In the cell-mediated assay, both DBC and MeDBC induced cytotoxicity and mutations in the target XP cells in a dose-dependent manner. However, DBC was effective at 2-fold lower concentrations than MeDBC. DNA adduct analysis using a 32P-postlabeling assay showed that at biologically significant low doses DBC was 2.5 times more effective than MeDBC in covalent binding to DNA. At higher doses, the difference in ability to bind to DNA was 1.3-fold. In both XP and Hs703T cells, DBC produced three major adducts, while MeDBC produced two major adducts, one of which was the same as one detected in the DBC adduct pattern. The number of DBC- and MeDBC-induced DNA adducts corresponding to a particular level of cytotoxicity and mutagenicity in the XP cells was 10 times lower than that for (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.

Comparative DNA binding of 7,12-dimethylbenz[a]anthracene and some of its metabolites in mouse epidermis in vivo as revealed by the 32P-postlabeling technique.

The binding of some mouse skin metabolites and related derivatives of the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) was investigated by 32P-postlabeling analysis after its topical administration. DMBA and trans-3,4-dihydro-3,4-dihydroxy-DMBA (DMBA-3,4-dihydrodiol) both led to the formation of four DNA adducts, which showed a very similar pattern of spots on thin-layer chromatograms. With trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA-8,9-dihydrodiol) one major adduct was obtained which was chromatographically indistinguishable from one of the DMBA adducts. In contrast, 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA) gave rise to two major adducts which were separable from DMBA adducts. 3-hydroxy-7,12-dimethylbenz[a]anthracene (3-OH-DMBA) and 7,12-dimethylbenz[a]anthracene-7,12-epoxide (DMBA-O2) did not lead to detectable amounts of adducts. Quantitative determination of DNA binding showed that an initiating dose (i = 100 nmol) of DMBA yielded approximately 12 adducts/10(7) normal nucleotides. Adduct formation with the same dose of DMBA-3,4-dihydrodiol was 7-8 times higher. At a 4-fold higher dose level, DMBA-8,9-dihydrodiol exhibited a 3- to 6-times weaker binding and 7-OHM-12-MBA a slightly stronger binding than DMBA. Chromatography of the DMBA and DMBA-3,4-dihydrodiol adducts with a solvent containing borate showed a decreased mobility of two out of four adducts in each case. These adducts were also sensitive to oxidation by periodate. The results suggest that two DMBA adducts carried vicinal cis-hydroxyl groups and thus were probably derived from the anti-3,4-dihydrodiol-1,2-oxide(s) of DMBA. The other two adducts were probably derived from the syn-stereoisomer(s). When the DNA-modifying capabilities and initiating activities of the more prominent mouse-skin metabolites are considered in relation to DMBA, DMBA-3,4-dihydrodiol is postulated to be a proximate and DMBA-3,4-dihydrodiol-1,2-oxide(s) to be ultimate initiators.

Postlabeling methods for carcinogen-DNA adduct analysis.

Radioactive carcinogens have provided most of our present knowledge about the chemistry of interactions between carcinogens and biological systems. The requirement of radioactive carcinogens has restricted carcinogen-DNA binding studies to chemicals that are readily available in isotopically labeled form, i.e., a minute fraction of all potentially mutagenic or carcinogenic chemicals. To extend the scope of carcinogen-DNA binding studies, an alternative method, which does not require radioactive test chemicals, has been developed. In this approach, radioactivity (32P) is being incorporated into DNA constituents by polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP after exposure of the DNA in vitro or in vivo to a nonradioactive, covalently binding chemical, and evidence for the alteration of DNA nucleotides is provided by the appearance of extra spots on autoradiograms of thin-layer chromatograms of digests of the chemically modified DNA. Quantitation of adduct levels is accomplished by scintillation counting. The sensitivity of the technique depends on the experimental conditions for 32P-labeling and on the chemical structure of the adducts. Greater sensitivity may be achieved if adducts can be separated as a class from the normal nucleotides. This is the case for an estimated 80% of all carcinogens, giving rise to bulky and/or aromatic substituents in DNA. Under the present conditions, one such adduct in 10(9) to 10(10) normal nucleotides can be detected. A total of approximately 80 compounds has been studied thus far Binding to DNA of rodent tissues was readily detected by the 32P-postlabeling assay for all known carcinogens among these compounds, and adducts were detected in DNA from human placenta of smokers.

Tissue-specific DNA adduct formation in mice treated with the environmental carcinogen, 7H-dibenzo[c,g]carbazole.

Covalent adduction of DNA by chemical agents is commonly thought to be an essential part of the initiation of chemical carcinogenesis. Until recently, assays of DNA damage by covalent binding of chemicals have been restricted mostly to substances that are available in radiolabeled form, which excludes many environmental compounds with carcinogenic potential. In this paper, the binding of non-radioactive 7H-dibenzo[c,g]carbazole (DBC), a known environmental carcinogen, to DNA in female CD-1 mice after s.c. injection of 44 mumol/kg of the compound has been investigated using a 32P-postlabeling assay. DBC showed strong hepatic specificity with a mean total level of 107 adducts per 10(7) nucleotides at 24 h, while much lower levels of binding were seen in kidney, lung, spleen, skin and brain with 4.3, 2.1, 1.3, 0.4 and 0.04 adducts, respectively, per 10(7) nucleotides. Proportions of individual DBC adducts also varied considerably between tissues. The degree of hepatic preference displayed by DBC is not seen with other polycyclic aromatic carcinogens such as benzo[a]pyrene and 2-acetylaminofluorene. The DNA-binding data, together with other hepatotoxic effects of the compound, may be causally related to the known hepatocarcinogenicity of DBC.