Combretastatin A-4 resistance in H460 human lung carcinoma demonstrates distinctive alterations in beta-tubulin isotype expression.

Tubulin isotype distribution may play a role in the development of anti-cancer anti-tubulin drug resistance as well as in drug efficacy and specificity. Stepwise selection was used to establish non-small cell lung carcinoma (NSCLC) H460 cells resistant to combretastatin A-4 (CA4), paclitaxel or vinblastine. The results demonstrated that the rate of CA4 drug resistance development was slower than that for paclitaxel. Western analysis demonstrated alterations in total beta-tubulin and classes I, III and IV tubulin isotypes among the resistant H460 cell lines. Class III beta-tubulin was significantly altered in all resistant cell lines. Cells resistant to paclitaxel, a structural stabilizer of microtubules, exhibited an increased expression while cells resistant to CA-4 and vinblastine, structural destabilizers of tubulin, demonstrated a reduction of the same isotype. To our knowledge, this is the first demonstration of resistance development and of the corresponding tubulin isotype response for the combretastatins.

Characterization of oncogene-silenced transgenic plants: implications for Agrobacterium biology and post-transcriptional gene silencing.

SUMMARY Agrobacterium tumefaciens tumorigenesis is initiated by the horizontal transfer of a suite of oncogenes that alter hormone synthesis and sensitivity in infected plant cells. Transgenic plants silenced for the iaaM and ipt oncogenes are highly recalcitrant to tumorigenesis, and present a unique resource to elucidate fundamental questions related to Agrobacterium biology and post-transcriptional gene silencing (PTGS). The oncogene-silenced transgenic tomato line 01/6 was used to characterize A. tumefaciens growth in planta and to screen for iaaM and ipt sequence variants. Even in the absence of macroscopic and microscopic indications of tumorigenesis, A. tumefaciens is capable of long-term survival in the hypocotyl tissues of the 01/6 line. A. tumefaciens growth, however, is significantly reduced in the 01/6 line, with populations decreased by 96% relative to wild-type at 52 days post-inoculation. In addition, the 01/6 line displayed suppression of tumorigenesis against all 35 tested strains of A. tumefaciens. High target homology is an absolute requirement of PTGS, therefore this result suggests that regions of the iaaM and ipt oncogenes are very highly conserved across most A. tumefaciens strains. Finally, graft transmissibility of oncogene silencing was assessed by grafting various non-silenced tomato genotypes on to the 01/6 line. Phenotypic and molecular evidence (tumorigenesis and absence of small interfering RNAs, respectively) suggest that oncogene silencing is not graft-transmissible, at least to wild-type and antisense iaaM-over-expressing genotypes.

A new technique for studying the stylet tracks of homopteran insects in hand-sectioned plant tissue using light or epifluorescence microscopy.

Homopteran insects, such as aphids, psyllids and scales, inject a proteinaceous salivary sheath into their host plant tissue during feeding. This sheath, also referred to as a stylet track, remains in the tissue after the stylets are withdrawn, and is useful for studying plant resistance to insects and plant virus transmission. We describe a new method for studying stylet tracks. Hand microtome sectioned plant material was fixed and cleared in ethanol. The stylet tracks were stained with acid fuchsin and counterstained with aniline blue or fast green. The acid fuchsin stained stylet tracks were pink to red under light microscopy, and orange under TRITC epifluorescence. Stylet tracks in unstained sections autofluoresced under DAPI epifluorescence. This new technique is significantly faster and less complex than previous techniques, and permitted visualization of stylet tracks with light or epifluorescence microscopy within 1 hr of collecting fresh plant material. The technique was also applicable to a broad range of homopterans and plant taxa and provided excellent photomicrographs.

Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin.

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.

Endocarp Dehiscence in Pistachio (Pistacia vera L.).

Two sites exist for endocarp dehiscence (shell split) in the pistachio nut (Pistacia vera L., Anacardiaceae). Longitudinal dehiscence occurs along lines of cells that have highly lignified, interlocking walls and are differentiated from cells of the neighboring endocarp only by being somewhat smaller and slightly elongate in the radial plane. Apical dehiscence occurs at the site where transmitting tissue penetrates the ovary wall to enter the ovarian locule. In most fruits, cells delineating the line of apical dehiscence possess thin, primary walls that stain only diffusely for pectic materials. In rare cases, possibly when apical dehiscence fails, these cells become lignified. Measurements of seeds and inner dimensions of endocarps of mature fruits show that dehiscent endocarps differ from indehiscent endocarps by having more massive seeds and seeds that are larger relative to the size of the endocarp. These results and observations indicate to us that two dehiscence events occur: longitudinal shell split, which is driven by physical forces exerted on the endocarp by the seed, and apical dehiscence, which occurs at the site of intrusion of the transmitting tissue tract into the ovary and varies according to the extent of transmitting tissue present at anthesis as well as the degree of cytodifferentiation that may occur in these cells.

A new anti-tubulin agent containing the benzo[b]thiophene ring system.

A new type of inhibitor of tubulin polymerization was discovered based on the 3-aroyl-2-arylbenzo[b]thiophene molecular skeleton. The lead compound in this series, 2-(4'-methoxyphenyl)-3-(3',4',5'-trimethoxybenzoyl)-6-methoxybe nzo[b]thiophene 1, inhibited tubulin polymerization, caused an increase in the mitotic index of CA46 Burkitt lymphoma cells, and inhibited the growth of several human cancer cell lines.

Molecular structures, conformational analysis, and preferential modes of binding of 3-aroyl-2-arylbenzo[b]thiophene estrogen receptor ligands: LY117018 and aryl azide photoaffinity labeling analogs.

Structural and computational modeling studies were performed on the antiestrogen LY117018 (3) and two photoaffinity labeling analogs, in which an azide replaces the basic ether side chain (methyl ether tetrafluoro azide 7 and its protio analog 8). These studies were undertaken in order to determine the conformational preferences of these compounds and to propose favorable orientational modes for their binding to the estrogen receptor. In the crystallographic studies, we found that, unlike tetrafluoro azide 7, which adopts a face-to-face stacking of the p-hydroxyphenyl and benzoyl groups in the solid state, the pendant rings in the corresponding protio analog 8 are found in a predominantly offset pi-stacked array. In LY117018, which has an ether on the benzoyl ring, stacking of the pendant rings does not occur in the crystal structure; it assumes a T-shape, with the benzoyl group oriented perpendicular to the benzo[b]thiophene nucleus. In modeling studies, analogs of LY117018, 7, and 8 were subjected to a conformational grid search by molecular mechanics, and for each compound, three low-energy conformers (and their atropisomers) were obtained. These conformers were further geometry optimized by semiempirical molecular orbital calculations. For each compound, one of the three minimum-energy conformers is quite similar to the solid-state geometry. The computational structure of the tetrafluoro azide showed the greatest stacking between the benzoyl group and the p-methoxyphenyl ring, but less stacking than was observed in the crystallographic structure. The orientational preferences of these benzo[b]thiophene ligands with the estrogen receptor were analyzed with the receptor volume mapping technique, a method based on the correspondence of the hydroxyl groups and the volume that the benzo[b]thiophene compound shares with a composite molecular volume of high-affinity estradiol-type ligands (the receptor excluded volume, RExV). If the benzo[b]thiophene nucleus is overlapped with the steroid AB rings, the best overlap with the RExV is achieved, but there is poor correspondence of the hydroxyl groups. An orientation and conformation in which the benzoyl group of the 3-benzoyl-2-aryl-benzo[b]thiophenes occupies a 7 alpha-like position relative to the steroid produces both ample volume overlap with the RExV and close approximation of the hydroxyl groups and is presented as the putative bioactive conformation.

Nonsteroidal estrogens bearing acyl azide functions: potential electrophilic and photoaffinity labeling agents for the estrogen receptor.

In an effort to develop novel affinity labeling agents for the estrogen receptor, we have synthesized two nonsteroidal ligands, a 1-aroyl-2-aryl tetralin system (1) and a 2-aryl-3-aroylbenzo[b]thiophene system (2). These agents, patterned after the Lilly antiestrogens trioxifene and LY 117018, respectively, embody acyl azide functions as part of a benzoyl chromophore. The acyl azide group has weak acylating activity, suitable for electrophilic affinity labeling, but this function is also photoreactive and, in its particular embodiment within these ligands, it could provide an efficient photochemical route to the highly reactive singlet acyl nitrene. The tetralin system (1) was prepared in nine steps from 6-methoxy-1-tetralone, and the benzothiophene system (2) was prepared in four steps from a known substituted benzo[b]thiophene precursor. In competitive binding assays, both compounds show reasonable binding affinity for the rat and lamb uterine estrogen receptor: estradiol = 100%, 1 = 3%, and 2 = 12%. When assayed by indirect receptor consumption assays, both compounds appear to have substantial capacity for irreversible binding (electrophilic reaction) with the receptor. This reactivity, which suggests that acylation of the receptor has occurred, is photoreversible. The nature of this ligand-receptor interaction is being investigated further.

Efficient and selective photoaffinity labeling of the estrogen receptor using two nonsteroidal ligands that embody aryl azide or tetrafluoroaryl azide photoreactive functions.

3-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-6-hydroxy-2-(4- hydroxyphenyl)benzo[b]thiophene 1 (tetrafluoroaryl azide, TFAA) and its protio analogue 3-(4-azidobenzoyl)-6- hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene 2 (protioaryl azide, PAA), photoaffinity labeling (PAL) reagents for the estrogen receptor (ER), have been prepared in high specific activity tritium-labeled form (19 Ci/mmol) and shown to undergo selective and efficient photocovalent attachment to ER from rat uterus. Both azides 1 and 2 demonstrate high binding affinity for ER as determined by both a competitive binding assay (relative binding affinities: estradiol = 100; TFAA = 9.3; PAA = 66) and a direct binding assay (Kd: estradiol = 0.24 nM; TFAA = 2.64 nM; PAA = 0.37 nM). When unlabeled TFAA and PAA are irradiated at greater than 315 nm, they demonstrate site-specific photoinactivation of ER that reaches 43% and 55%, respectively, by 30 min. Specific photocovalent attachment to ER can be effected by irradiation of the tritium-labeled azides; the covalent attachment efficiency is good (1 = 20-30%, 2 = ca. 25%) and the selectivity of ER labeling is high. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of a major component at Mr 60,000 and a minor species at Mr 46,000, the same two species that are labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. The ER-specific antibodies H222Sp gamma and D547Sp gamma show a clean precipitation of only these two species. In the MCF-7 human breast cancer cell line, PAA is a full estrogen agonist in terms of stimulation of cell proliferation and induction of progesterone receptor. These two azides provide the first system in which the photocovalent attachment efficiency of an aryl azide can be compared to its tetrafluorosubstituted aryl azide analogue in a complex biological receptor system. Azides 1 and 2 are the most efficient and selective PAL reagents prepared to date for ER, and they should be useful in further studies of the hormone-binding domain of this protein.

Torsionally and hydrophobically modified 2,3-diarylindenes as estrogen receptor ligands.

2,3-Diarylindenes are ligands for the estrogen receptor which display intrinsic fluorescence. In order to optimize the receptor binding affinity of these compounds while preserving their desirable fluorescence properties, a series of torsionally modified analogues were prepared. A fluorine or methyl group was introduced on either of the two phenyl substituents ortho to their attachment site to the indene nucleus, in order to increase the out-of-plane twist of the appended rings. The analogues were prepared by the benzylation of appropriate deoxybenzoins, followed by Friedel-Crafts cyclic alkylation-dehydration. Comparison of the X-ray crystal structure of one analogue with unsubstituted analogues confirms the torsional perturbation effected by the ortho substituent. The torsional disposition of the C-2 aryl group in the substituted diphenylindenes is further investigated by UV (absorbance maxima and molar absorptivities), fluorescence (Stokes' shift), and NMR (chemical shifts). These spectroscopic measurements indicate increasing twisting between the C-2 aryl substituent and the indene system according to the following order: 3-ring o-Me-indene 9f less than diphenylindene 15 = 20 degrees less than 3-ring o-F-indene 9c less than 1-Me-indene 16 less than 2-ring o-F-indene 9b less than 2-ring o-Me-indene 9e = 63 degrees. The binding affinity of these analogues to the estrogen receptor was evaluated by a competitive radiometric receptor binding assay. While o-fluoro or o-methyl substitution on the 3-ring increases binding only slightly, binding of the o-fluoro 2-ring analogue is increased ca. 6-fold and the o-methyl analogue 11-fold, giving, in the latter case, a compound with an affinity equivalent to that of estradiol. The increase in binding affinity afforded by ortho substitution correlates with the increase in the torsion angle of the C-2 aryl ring. A thermodynamic evaluation of the receptor fit (Andrews, P. R.; Craik, D. J.; Martin, J. L. J. Med. Chem. 1984, 27, 1648) indicates that, for the o-methyl 2-ring analogue, the effect of the ortho substitution on increasing receptor binding appears to be a combination of increased surface area due to the substituent itself, together with a change in surface area of the ligand that results from the increased torsion of the two aryl rings. An o-fluoro substituent on the 2-ring provides a compromise between the relative planarity required for high fluorescence intensity and the molecular shape needed for increased estrogen receptor binding affinity.(ABSTRACT TRUNCATED AT 400 WORDS)

[3H]DU41165: a high affinity ligand and novel photoaffinity labeling reagent for the progesterone receptor.

17 alpha-Acetoxy-6-fluoro-16-methylene-(9 beta, 10 alpha)pregna-4,6-dien- 3,20-dione (DU41165), a retroprogestin (9 beta, 10 alpha) embodying a fluorine-substituted dienone system, has been prepared in high specific activity tritium-labeled form (4 Ci/mmol) and shown to be a high affinity ligand for the progesterone receptor (PgR) and a highly selective photoaffinity labeling reagent for PgR. The radiosynthesis involved conversion of DU41231 (the 17 alpha-hydroxy analog of DU41165) to DU41165 by treatment with tritium-labeled acetic anhydride. The binding affinity of DU41165 for PgR was determined by both a competitive binding assay and a direct binding assay (Scatchard analysis) to be 1.6-2.2-times higher than that of the high affinity synthetic progestin promegestone (R5020). In unlabeled form, DU41165 demonstrates photoinactivation of PgR to the extent of 60% at 60 min. In radiolabeled form [3H]DU41165 demonstrates specific covalent attachment with an efficiency of 5-7%. SDS-polyacrylamide gel electrophoresis of photoattached [3H]DU41165 confirms that there is covalent labeling of both the B subunit (Mr = 118,000), and the A subunit (Mr = 88,000) of PgR in a molar ratio of approximately 1:3.

Target tissue uptake selectivity of three fluorine-substituted progestins: potential imaging agents for receptor-positive breast tumors.

We have studied three new fluorine-substituted progestins (1-3) as potential imaging agents for progesterone receptor (PgR)-positive human breast tumors. Two of these are fluorine-substituted analogs of the potent progestin R5020 (promegestone), derived from (21S)-hydroxy R 5020 (RU 27987) and (21R)-hydroxy R 5020 (RU 27988), known metabolites of R 5020, which have affinities for PgR that are 116 and 4%, respectively (relative to R 5020 = 100%). These precursors were protected as their 3,3-dioxolane derivatives and converted to the 21-trifluoromethanesulfonate derivatives. Fluoride ion displacement, followed by acid-catalyzed deprotection, furnished in good yield the epimeric fluoroanalogs, (21S)- and (21R)-fluro R 5020 (1 and 2, affinities for PgR, 11 and 45%, respectively). These compounds were also prepared in 18F labeled form by the same route, in 14-32% overall radiochemical yield (decay corrected; synthesis time 90 min; sp. act. 370-1060 Ci/mmol). In tissue distribution studies in estrogen-primed immature rats, uterus-to-muscle ratios were 4.3 at 1 h for the 21S-epimer and 1.1 for the 21R-epimer, paralleling their relative binding affinities. Considerable metabolic defluorination was observed. The third fluorine-substituted progestin, DU 41165, has a novel retroprogesterone (9 beta, 10 alpha) structure, substituted with fluorine at C-6; its binding affinity is 145% relative to R 5020, and it was prepared in tritium-labeled form by acetylation of DU 41231, the 17 alpha-hydroxy precursor, with [3H]acetic anhydride. In estrogen-primed immature rats, this compound shows uterus-to-muscle ratios of 15 at 1 h, and 18-71 between 2 and 6 h, suggesting that compounds in this retroprogesterone series may be very promising candidates for selective imaging of PgR-positive tissues and tumors.

Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation.

Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants.