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PubMed1990

Phosphorylation of the estradiol receptor in MCF-7 human breast cancer cells in culture.

Double labelling and Western blot techniques were used to demonstrate phosphorylation of estradiol receptor. Cells in monolayer culture were incubated with [32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine [( 3H]TA). Labelled receptor was precipitated with the monoclonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose, and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or transferred to nitrocellulose paper. Receptor protein was detected on the Western blot with the monoclonal antibody H222 and rabbit anti-rat peroxidase conjugate. Phosphorylated receptor was visualized by autoradiography. Tritium and 32P activities were monitored in the gels. Two phosphorylated forms of the receptor (molecular weights 67 and 50 kDa) have been detected in MCF-7 cells. Estradiol treatment of the cells was found to increase phosphorylation of the receptor. In estradiol-treated cells both phosphorylated receptor forms were present mainly in the nuclear extract. Both forms bound [3H]TA as evidence by SDS-PAGE. [3H]TA binding was abolished by excess non-radioactive estradiol. In addition two phosphorylated proteins of approximately 120 and 90 kDa were regularly coprecipitated with receptor in cytosol. These proteins did not bind [3H]TA. The 90 kDa phosphorylated protein was identified as a heat shock protein (hsp-90).

Blotting, Western↗

PubMed1990

Estradiol increases phosphorylation of the 90 kDa heat shock protein not associated with estradiol receptor in MCF-7 cells in culture.

MCF-7 cells in monolayer culture were incubated with [32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine [( 3H]TA). The heat shock protein (hsp-90) bound to receptor was precipitated with monoclonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Hsp-90 not associated with receptor was similarly purified after isolation with the monoclonal antibody AC88. It was found that estradiol treatment of the cells markedly increased phosphate incorporation in the free hsp-90, without affecting heat shock protein bound to receptor. A 6-fold increase in phosphate content was observed after 10 min incubation of the cells with estradiol. A similar effect was seen after treatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). The calcium ionophore A23187 had no influence on hsp-90 phosphorylation, and treatment of the cells with forskolin to increase the cellular content of cAMP had a reverse effect. A 50% reduction of the phosphate content in the free hsp-90 was observed after 15 min treatment. The observation that estradiol, TPA and forskolin had effect only on hsp-90 not bound to receptor is an indication that the receptor-hsp-90 complex exists in vivo. Time course studies show that the effect of estradiol is non-genomic. Two possible explanations of the results seem to exist. Either estradiol induces an increase in the degree of phosphorylation of hsp-90, or hsp-90 is translocated to the cytosol from a different cellular compartment.

Blotting, Western↗

PubMed1993

Characteristics of a membrane-associated antilactogen binding site for tamoxifen.

The antilactogen binding site (ALBS) is a membrane associated protein to which tamoxifen (TAM) and related non-steroidal antiestrogens, but not estrogen, bind. It is through this site that TAM inhibits lactogen binding to the prolactin (Prl) receptor and subsequent Prl induced growth and differentiation in target tissues. Binding of lactogens to the Prl receptor is inhibited by TAM or 4-hydroxy-TAM at 4 degrees C as well as room temperature, thus suggesting that the ALBS is not an enzyme. TAM acts by inhibiting the binding of lactogens to the receptor rather than promoting dissociation of the hormone-receptor complex. Lactogens bind to mammary gland membranes with an Kd of 4.3-8.2 x 10(-10) M. In the presence of 10(-7) M TAM the affinity decreased to a Kd of 0.8-1.6 x 10(-9) M. Binding of 3H-TAM to mammary gland membranes was effectively inhibited by an anti-Prl receptor antibody, thus suggesting a close relationship between the Prl receptor and the ALBS. Separate affinity purification of the ALBS and the Prl receptor resulted in peak fractions demonstrating specific binding activity for both TAM and lactogenic hormones. Re-isolation of the affinity purified Prl receptor on a TAM-Sepharose affinity resin again resulted in co-elution of both binding activities. The isolates from both affinity resins contained primarily a single band with an apparent molecular mass of 90 kDa. This band was precipitated with the anti-Prl receptor antibody and specifically bound the affinity label ring-3H-TAM aziridine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

In the search for new anticancer drugs. XX: A comparison of the in vitro growth inhibition of P388 cells by TEPA and N,N;N',N'-bis (1,2-ethanediyl)-N''-(1-oxyl-2,2,6,6-tetramethyl-4- piperidinylaminocarbonyl) phosphoric triamide and congeners.

We tested the in vitro growth inhibitory activity of TEPA, and three analogs against P388 murine lymphocytic leukemia cells in culture. The analogs consist of spin-labeled TEPA and two reduced forms containing the NH and NOH groups instead of the nitroxyl function. Spin label TEPA was obtained by replacing one of the aziridine groups in TEPA with spin-labeled urea. In a concentration range of 10(-6)-10(-5) M, only the reduced analog containing the NH group was active. That is, to achieve a 50% inhibition of cell growth, a five-fold excess in concentration of this analog (IC50 = 10 X 10(-6) M) was needed as compared to the parent compound TEPA (IC50 = 2 X 10(-6) M). These results are in contrast with those obtained in vivo against the same leukemia cell line, indicating inherent discrepancies between in vivo and in vitro evaluation of antitumor agents.

Animals↗

PubMed1989

Radiosensitizing and cytotoxic effects of nitroimidazoles in CHO cells expressing elevated levels of glutathione-S-transferase.

A cell line, CHO-Chlr, which has elevated levels of glutathione-S-transferase (GST), is resistant to damage caused by bi-functional nitrogen mustards. This cell line has a similar radiation sensitivity to and value of oxygen enhancement ratio as the wild type cells, CHO-K1, from which the mutant line was derived. Hypoxic cell radiosensitizing efficiencies for misonidazole, etanidazole (SR2508), and pimonidazole (R0 03-8799) are similar in both cell lines with values for C1.6 of 0.5, 0.75, and 0.08 mmol dm-3 for each drug respectively. In contrast, for RSU 1069 the sensitizing efficiency is lower in the cell line showing elevated levels of GST. The hypoxic toxicity of RSU 1069 and misonidazole is similar in each cell line, whereas in air, toxicity is 2 fold less in the CHO-Chlr cells. These results suggest that reaction of the alkylating aziridine part of RSU 1069 contributes to the high sensitizing efficiency of this drug. This is likely to be a consequence of either binding of the sensitizing moiety at the target site (ie, increased local concentration or induction of sub-lethal damage that is only expressed on subsequent irradiation.

Animals↗

PubMed1994

In vitro cytotoxicity and chemosensitizing activity of the dual function nitroimidazole RB 6145.

PURPOSE: To determine the cytotoxicity and chemosensitizing potential of RG 6145 in mouse KHT/iv and human A549 tumor cells. METHODS AND MATERIALS: RSU 1069, the lead compound in a series of nitroimidazoles containing an alkylating aziridine function, has been shown to possess a high degree of selective cytotoxicity for hypoxic cells in addition to being a potent sensitizer of radiation and chemotherapy. Unfortunately, preliminary clinical studies have revealed a dose-limiting gastrointestinal toxicity for RSU 1069. Recently RB 6145, the ring-opened analogue of RSU 1069, has been found to be less emetic than RSU 1069. In the present studies, we assessed both the differential hypoxic cell cytotoxicity of RB 6145 and its chemosensitizing potential when combined concomitantly with variable doses of the activated form of cyclophosphamide (4-hydroperoxy-cyclophosphamide, 4-OOH-CP) or the nitrosourea CCNU. RESULTS: As we had observed previously for RSU 1069, RB 6145 was found to be less cytotoxic to human than rodent tumor cells. In addition, the degree of selective cytotoxicity toward hypoxic cells was (a) less in A549 than in KHT/iv cells (factor of 9 vs. 80) but (b) comparable to that seen with RSU 1069. For both cell lines, inclusion of the sensitizer enhanced the cell killing of the chemotherapeutic agent 4-OOH-CP by a factor of approximately 1.5-1.7-fold. When combined with CCNU, RB 6145 increased the killing of A549 cells approximately 1.8-fold. Similar hypoxic cell preferential cytotoxicity and enhancement in anti-tumor treatment efficacy were seen when A549 cells were exposed to the R enantiomer of RB 6145 (PD 144872) either alone or in combination with CCNU. CONCLUSION: These data support the notion that further consideration should be given to the clinical application of these bioreductive agents.

Animals↗

PubMed1996

Inducible synthesis of the mitomycin C resistance gene product (MCRA) from Streptomyces lavendulae.

The mcr locus from Streptomyces lavendulae confers high level resistance (> 100 micrograms/ml) to mitomycin C (MC) and related mitomycins when cloned into Streptomyces lividans. Production of the mcrA gene product (MCRA) was shown to be MC-inducible by identification of MCRA (M(r) of 54 kDa) using Western blot analysis and enzyme linked immunosorbent assay (ELISA). The magnitude of MCRA production was dependent on MC concentration, with primary induction starting at 0.1 microgram/ml and maximum induction at 10 micrograms/ml of the drug. Different levels of MCRA production were observed when other mitomycin metabolites were used as inducers, and the level of induction related directly to aziridine ring substitution on the individual molecules. Moreover, inducible synthesis of the mcr A gene product was unique to this structural class since production of MCRA did not occur as a general response to DNA damaging agents. The time profile of intracellular MCRA synthesis correlated with MC production in S. lavendulae, suggesting coordinated regulation of MC resistance and biosynthetic genes.

Antibiotics, Antineoplastic↗

PubMed1994

Derivatization of thiol-containing compounds.

The determination of thiol-containing compounds in biological fluids is important in biochemistry and clinical chemistry. In this paper, derivatization reagents for thiols are reviewed with respect to their reactivity, selectivity, spectroscopic characteristics and their applicability especially to high-performance liquid chromatography. Derivatization used in ultraviolet and electrochemical detection. The derivatization reagents contain a functional group, e.g. an N-substituted maleimide, active halogen or aziridine, which react with the thiol group. Derivatization for use in flow injection analysis, thin-layer chromatography or gas chromatography-mass spectrometry is also described.

Chromatography, High Pressure Liquid↗

PubMed1992

Accumulation of a non-binding form of estrogen receptor in MCF-7 cells under hydroxytamoxifen treatment.

It is well known that MCF-7 cells, when incubated with hydroxytamoxifen (OH-Tam) loose their capacity to bind [3H]estradiol. By using Western blotting and [3H]tamoxifen aziridine labeling of KCl extracts from these cells we found that this loss in binding capacity was not associated with a disappearance of the estrogen receptor (ER) protein, an event known to occur after incubation with estradiol. Attempts to label under exchange conditions these ER molecules, which, on the basis of enzyme immunoassays appear to accumulate under OH-Tam treatment, were unsuccessful. Cell fractionation suggested that their origin is nuclear. Assessment of a few triphenylethylenic antiestrogens, as far as their inhibitory potency towards the in vitro MCF-7 cell growth is concerned, indicated a correlation between accumulation of these non-binding ER molecules and the antiestrogen antiproliferative action. However, we were unable to demonstrate absence of such an ER accumulation in two tamoxifen-resistant variants. Impaired folding of the ER protein or impaired phosphorylation of its hormone-binding domain are attractive hypotheses to account for these non-binding ER molecules. Whether these ER molecules have any physiological role, such as competition with the "normal" receptor molecules for the estrogen responsive elements on the DNA is unknown and deserves further study.

Blotting, Western↗

PubMed1993

An assessment of the role of domain F and PEST sequences in estrogen receptor half-life and bioactivity.

The estrogen receptor (ER) is a rapidly turning over protein, with a half-life of ca. 3-4 h in estrogen target cells. Sequence analysis of the human ER reveals a putative PEST sequence, sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy-terminal F domain of the protein. Since PEST sequences have been implicated in the rapid turnover of some proteins, we have used site-directed mutagenesis to investigate the role of the F region containing PEST residues in the stability and bioactivity of the receptor. A truncated form of ER lacking the last 41 amino acids of the protein and encompassing the PEST sequences (amino acids 555 to 567) was made by mutagenesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipitation of [35S]methionine/[35S]cysteine labeled receptors or of receptors covalently labeled with tamoxifen aziridine followed by gel electrophoresis, were used to determine the half-life of the wild-type and truncated ERs. These experiments showed that the turnover rate of the receptors expressed in Chinese hamster ovary and monkey kidney (COS-1) cells was 3 to 5 h and that elimination of the PEST residues did not have a significant effect on the degradation rate of the protein. Moreover, deletion of the last 41 amino acids (F domain) of the ER did not affect transactivation ability, ligand binding affinity, or the phosphorylation pattern of the receptor. Therefore, the role of domain F in ER function remains unclear, but it is not a determinant of the relatively rapid rate of ER turnover in cells.

Algorithms↗

PubMed1996

Estrogenic and antiestrogenic regulation of the half-life of covalently labeled estrogen receptor in MCF-7 breast cancer cells.

Effect of estrogens and antiestrogens (AEs) on estrogen receptor (ER) half-life was analyzed in MCF-7 cells by assessing its progressive disappearance after covalent labeling in situ with [3H]tamoxifen aziridine ([3H]TAZ). Cells were incubated for 1 h with 20 nM [3H]TAZ either in the absence or presence of a 500-fold excess of unlabeled estradiol (E2) (non-specific binding). The entire ER population was labeled by this method as established by subsequent incubation of the cells with [125I]E2. [3H]TAZ labeled cells were maintained in culture for additional 5 h in the absence (control) or presence of increasing amounts (0.1 nM - 1 microM) of either a given estrogen (E2, estrone, diethylstilbestrol, bisphenol), a pure AE (RU 58 668, ICI 164 384) or an AE with residual estrogenic activity (RU 39 411, 4-hydroxytamoxifen, keoxifene). The progressive disappearance of nuclear and cytosolic [3H]TAZ-ER complex during 5 h incubation were assessed by their immunoprecipitation with anti-ER monoclonal antibody (H 222) followed by scintillation counting or SDS-PAGE and fluorography. Fading of labeled receptors was extremely slow (approximately 10% loss after 6 h) in absence of any hormone/antihormone indicating a long half-life of the [3H]TAZ-ER complex. Addition of estrogens as well as pure AEs led to a dramatic reduction of the half-life while AEs with residual estrogenic activity were extremely less efficient in this regard providing an explanation for the ability of latter compounds to up-regulate the receptor since they do not affect ER mRNA synthesis and stability. Receptor disappearance induced by estrogens was closely related to their binding affinity for ER. Newly synthesized ER emerged during the treatment with hormones or antihormones seems to be implicated in the phenomenon since [3H]TAZ was covalently bound and could, therefore, not be displaced by these compounds. Induction of synthesis of a short half-life peptide(s) with degradative activity was demonstrated by addition of cycloheximide or puromycine (both at 50 microM) which completely blocked ER disappearance. The fact that no cleavage products of ER were detected by SDS-PAGE suggested a lysosomial hydrolysis. Hence, hormonal modulation of only a part of ERs may down-regulate their total population until it reaches the steady-state level.

Breast Neoplasms↗

PubMed1996

Footprint analysis of estrogen receptor binding to adjacent estrogen response elements.

Quantitative DNase I footprinting assays were employed to simultaneously measure the amount of estrogen receptor (ER) bound to each site in constructs containing multiple estrogen response elements (EREs). These assays revealed identical, high affinity ER-ERE binding, Kd of approximately 0.25 nM, for estradiol-liganded ER (E2-ER), 4-hydroxytamoxifen liganded ER (4-OHT-ER), tamoxifen aziridine liganded ER (TAz-ER), and unliganded dimeric ER, for each ERE in constructs containing up to four tandem EREs. Increasing concentrations of ER resulted in the same pattern of occupancy for each ERE, whether or not the site was located near other EREs. Similarly, the presence or absence of E2, 4-OHT, or TAz ligand did not change ER-ERE interaction. Since activated ER-ERE binding affinity is identical, whether ER is liganded or unliganded, ligand cannot regulate ER-ERE binding affinity. These results support the hypothesis that ligand-dependent conformational changes primarily determine how ER interacts with components of the transcription initiation complex that mediate gene transactivation. In addition, footprint assays revealed that, following ER binding, an AT-rich site adjacent to the ERE becomes hypersensitive to DNase I digestion. This sequence may be easily or intrinsically bent, assisting in recruiting ER to ERE sites.

Animals↗

PubMed2004

Novel O-glycosyl amino acid mimetics as building blocks for O-glycopeptides act as inhibitors of galactosidases.

As potential lead structures for a new class of glycosidase inhibitors the novel O-glycosyl amino acid mimetics 3'-O-[2,6-anhydro-D-glycero-L-gluco-heptitol-1-yl]-L-serine 3 and-L-threonine 4 were synthesized, employing regio- and stereoselective aziridine ring opening methodology. They proved to be stable in the presence of glycosidases and showed competitive inhibition of alpha-galactosidase from Aspergillus niger.

Amino Acids↗

PubMed2006

Total synthesis and biological evaluation of ustiloxin natural products and two analogs.

Synthetic investigations of ustiloxin natural products are described. The first total synthesis of ustiloxin F was completed in 15 steps via ethynyl aziridine ring-opening by a phenol derivative. The results of biological tests of synthetic ustiloxins D and F, and two analogs, O-Me-ustiloxin D and 6-Ile-ustiloxin, demonstrated that the free hydroxyl group ortho to the ether linkage is critical for activity and variations at the Val/Ala site produce changes in the biological activity suggesting the need for further perturbations at this site to more extensively study the tubulin binding.

Biological Products↗

PubMed2005

Synthetic studies on heterocyclic natural products.

This article reviews past and ongoing research in the author's laboratory directed toward the synthesis of natural products displaying an azaspirocyclic framework, or incorporating a medium-ring nitrogen heterocycle. New synthetic technologies were devised in order to address the synthetic problems posed by the target molecules. Thus, efforts in the area of azaspirocyclic substances have relied on an oxidative amidation of phenols promoted by iodobenzene diacetate, whereas access to medium-ring nitrogen heterocycles has been secured by means of a ring expansion sequence that relies on the fragmentation of an aziridine triggered by a homo-Brook transposition. Details of the development of these technologies are presented, together with applications to the total synthesis of FR-901483, TAN-1251C, cylindricines, and mitomycinoids.

Aza Compounds↗

PubMed2006

New impurity profiles of recent Australian imported 'ice': methamphetamine impurity profiling and the identification of (pseudo)ephedrine and Leuckart specific marker compounds.

Determination of the synthetic route in methamphetamine profiling relies on the identification of key, route specific marker impurity compounds. Impurity profiling of recent crystalline methamphetamine ('ice'), seized by the Australian Federal Police at the Australian border in 2003-2004, identified route specific marker compounds normally associated with two different synthetic approaches in the same samples. Impurities consistent with the ephedrine route included 1,2-dimethyl-3-phenyl aziridine (I), 1,3-dimethyl-2-phenylnaphthalene (VIII) and 1-benzyl-3-methylnaphthalene (IX), alongside N-formylmethamphetamine (V), N,N-di-(beta-phenylisopropyl)amine (XII) and N,N-di-(beta-phenylisopropyl)methylamine (XIII), N-benzoylamphetamine (XIV) and N,N-di-(beta-phenylisopropyl)formamide (XV) commonly associated with the Leuckart route and/or reductive amination. A non-synthetic process related origin of low-level N-formylmethamphetamine is proposed.

Journal Article↗

PubMed2005

LC-UV method development and validation for the investigational anticancer agent imexon and identification of its degradation products.

Imexon (4-imino-1,3-diazabicyclo[3,1,0]-hexan-2-one) is a member of the class of 2-cyanoaziridine derivatives, which have been of interest as immunomodulators and anticancer agents since the late 1970s. The pharmaceutical development of imexon necessitated the availability of an assay for the quantification and purity determination of imexon active pharmaceutical ingredient (API) and the drug in its pharmaceutical dosage form. A liquid chromatographic (LC) method with ultraviolet (UV) detection was developed, using a reverse phase column with phosphate buffer (pH 6; 50 mM) as mobile phase and UV detection at 230 nm. Although retention capacity for imexon was small (capacity factor of 0.5), the method was found to be linear over the concentration range of interest of 1.0-25 microg/mL, precise, accurate, and stability-indicating. Moreover, the use of LC-mass spectrometry (MS) and on-line photodiode array (PDA) detection enabled us to propose structures for four degradation products. Two of these products were also found as impurities in the API. The degradation products, including chloro- and hydroxy-derivatised products were shown to arise from nucleophilic reactions with the activated aziridine moiety of imexon. The developed LC-UV method was found suitable for the pharmaceutical quality control of imexon API and the drug in its pharmaceutical dosage form.

Antineoplastic Agents↗

PubMed1976

A synthesis of 3',4'-dideoxykanamycin B.

3',4',-Dideoxykanamycin B, the kanamycin B derivative that is active against resistant bacteria, was prepared from kanamycin B via N-tosylation, 3',4'-O-sulphonylation, 3',4'-unsaturation, and hydrogenation. The unsaturated intermediate was obtained from the 3',4'-di-O-sulphonyl derivatives by the action of sodium iodide in N,N-dimethylformamide; if zinc dust was added in this reaction, aziridine derivatives were formed. Removal of the tosyl group was successfully performed by using sodium in ammonia-ethylamine.

Dibekacin↗