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PubMed1997

The role of NAD(P)H:quinone oxidoreductase in quinone-mediated p21 induction in human colon carcinoma cells.

This study examines the role of NAD(P)H:quinone acceptor oxidoreductase (NQOR) (EC 1.6.99.2) in the metabolism of aziridinylbenzoquinones and the ensuing formation of reactive oxygen species in the induction of the cell cycle inhibitor p21 (WAF1, Cip1, or sdi1) in human colon carcinoma cells. The aziridinylbenzoquinones used were 2,5-diaziridinyl-1,4-benzoquinone (DZQ) and 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ). The cell lines used in this study, BE and HT29 human colon carcinoma cell lines, are devoid of and overexpress NQOR activity, respectively. The rate of reduction of the above quinones in BE cells proceeded at similar rates (approximately 170 nmol/min/ mg protein) and, expectedly, it was not affected by the NQOR inhibitor, dicumarol. The metabolism of DZQ in HT29 cells was largely accomplished by NQOR (approximately 94%), whereas that of AZQ was accomplished by dicumarol-insensitive reductases. The metabolism of DZQ in HT29 cells was accompanied by H2O2 formation, which was approximately 10-fold higher than that ensuing from the activation of AZQ. In agreement with these data, the production of H2O2 during the activation of DZQ by purified NQOR was approximately 10-fold higher than that of AZQ. The formation of H2O2 during the metabolism of aziridinylbenzoquinones in BE cells was 24- to 57-fold lower than that in HT29 cells. At variance with HT29 cells, H2O2 formation by BE cells was insensitive to the catalase inhibitor sodium azide. The bioactivation of AZQ and DZQ in BE cells yielded O2.- and HO. as detected by spin trapping/EPR, the intensity of the former adduct being approximately 2-fold higher than that of the latter. These signals were insensitive to dicumarol. The metabolism of DZQ in HT29 cells yielded mainly HO. and a modest contribution of O2.- (ratio HO./O2.- approximately 10), whereas that of AZQ yielded a HO./O2.- approximately 2. The effect of dicumarol on the free radical pattern obtained during DZQ metabolism resulted in a strong inhibition (80%) of HO. production and a substantial increase of O2.- generation. The metabolism of DZQ and AZQ in BE cells was associated with a significant increase of p21 mRNA levels; the former quinone was approximately 2-fold more efficient than the latter. DZQ metabolism in HT29 cells led to an increase of p21 mRNA levels 15-fold higher than that observed with AZQ activation. Dicumarol did not inhibit p21 induction associated with the metabolism of DZQ in the NQOR-deficient BE cells, whereas the inhibitor decreased p21 induction in HT29 cells by approximately 30%. This modest inhibition is likely due to the low concentration of dicumarol used, which did not affect p21 constitutive levels in control experiments carried out in the absence of the quinone. p21 induction in HT29 cells was also inhibited by DTPA, a metal chelator, and N-acetylcysteine, a potent cellular anti-oxidant, suggesting that HO. may serve as an ultimate mediator for the induction. It may be surmised that the higher efficiency of DZQ in p21 induction may be related to its efficient metabolism by NQOR in HT29 cells and the associated high level of reactive oxygen species. The role of reactive oxygen species in p21 induction was further assessed upon supplementation of cells with H2O2:p21 induction in BE cells was 4-fold higher than that in HT29 cells. These findings suggest that assessment of the role of NQOR and reactive oxygen species in p21 induction requires careful consideration of the cell genotype.

Acetylcysteine↗

PubMed1997

Synthesis of the photoaffinity label [1'-14C]-6C-(azimethyl)octylglucoside and its reaction with isolated renal brush border membranes.

We describe the synthesis of the photolabile analog of n-octylglucoside (OG), 6C-(azimethyl)octylglucoside (diazirino-octylglucoside, DOG). This diazirino derivative (lambda max = 335 nm) can be activated with long-wavelength UV to generate a highly reactive carbene, an intermediate suitable for a covalent ligand-receptor linkage. In inhibitor studies on D-glucose uptake into rabbit renal brush border membrane vesicles (BBMV) DOG showed a competitive inhibition with regard to D-glucose with a Ki of 20 +/- 5 microM, which was not significantly different from that of the template compound OG (Ki = 30 +/- 10 microM). On irradiation (lambda max = 350 nm) of BBMV in the presence of [14C]DOG, proteins of 61, 72 to 78, and 95 kDa were labeled by photoinduction. A band of 73 kDa was also positive in Western blots using an anti-Na+/D-glucose cotransporter antibody. Further investigations revealed that the labeled proteins were presumably mainly glucosidases known to be present in high concentration in renal BBMV. By comparison with authentic material one of the labeled proteins (61 kDa) was identified as trehalase (EC 3.2.1.28). These studies suggest that labels such as DOG carrying the reactive group directly at the D-glucose moiety of the molecule can be used to label proteins with carbohydrate recognition sites.

Affinity Labels↗

PubMed1999

Sample preparation for peptide mapping--A pharmaceutical quality-control perspective.

In quality control of therapeutic proteins peptide mapping is used for confirmation of primary structure and detection of posttranslational modifications. The demands put on the experimental procedure are therefore different than in the case of determination of an unknown protein structure. It is here recognized that a peptide-mapping method for quality control of proteins should be inert (not induce or revert modifications), general, robust, and allow a high sample throughput. The steps prior to the separation of the generated peptides are identified as crucial for meeting these demands. This includes denaturation, reduction, alkylation, buffer exchange, solubilization, and digestion. A critical review of the literature regarding these steps is presented. Relevant options in all steps are experimentally evaluated. Novel approaches are developed for many of the steps. The result is a sample preparation procedure that essentially meets the stated demands.

Alkylation↗

PubMed1995

Evaluation by polymerase chain reaction on the effect of beta-propiolactone and binary ethyleneimine on DNA.

Inactivating treatments for viruses such as pasteurization or alkylation by beta-propiolactone or binary ethyleneimine were tested for their capacity to modify nucleic acids. The modification of a nucleic acid was measured as the decrease in spot intensity in Southern blots after polymerase chain reaction (PCR) amplification. The inactivating treatments were applied to cellular and viral genomic material from a human lymphoblastoid cell line immortalized by Epstein Barr Virus (EBV), which produced a monoclonal antibody. Pasteurization did not modify the ability to amplify and detect cellular or viral DNA. Binary ethyleneimine strongly reduced the amount of detectable DNA and beta-propiolactone under particular conditions of incubation abolished all trace of DNA.

Alkylating Agents↗

PubMed1994

Vitamin E prevents the place learning deficit and the cholinergic hypofunction induced by AF64A.

The present study examined whether pretreatment with vitamin E would attenuate the place learning impairment and the neurochemical deficits induced by intracerebroventricular (icv) injection of the cholinotoxin ethylcholine mustard aziridinium ion (AF64A). Male Sprague-Dawley rats were pretreated by intramuscular injection of either vitamin E or saline 24 h and 15 min prior to surgery. They were then infused bilaterally into the cerebroventricles with AF64A (3 nmol/side) or artificial cerebrospinal fluid (CSF). Following 14 days of postoperative recovery, the rats were trained to acquire a place learning task in a water maze for two blocks of four trials a day for 5 days. Following the 40th trial a probe trial was used to assess memory for the platform location. After completion of behavioral testing, hippocampal high-affinity choline uptake (HAChU) was assessed. The groups which had received either saline or vitamin E pretreatment followed by icv injection of CSF did not differ significantly on any parameter measured and were therefore pooled as control group. Animals which had been pretreated with vitamin E and had received icv injection of AF64A exhibited neither significant impairment in water maze performance nor significant decrease in HAChU. In contrast, animals which had been pretreated by saline followed by icv injection of AF64A were significantly impaired in acquisition of the place learning task as well as during the probe trial and had reduced HAChU in the hippocampus. These findings indicate that vitamin E may have a neuroprotective effect in the septohippocampal cholinergic system.

Animals↗

PubMed2001

Combined adenovirus-mediated nitroreductase gene delivery and CB1954 treatment: a well-tolerated therapy for established solid tumors.

Gene-directed enzyme prodrug therapy (GDEPT) is a refinement of cancer chemotherapy that generates a potent cell-killing drug specifically in tumor cells by enzymatic activation of an inert prodrug. We describe in vivo studies that evaluate the efficacy and safety of intratumoral (i.t.) injection of an adenovirus vector (CTL102) expressing Escherichia coli nitroreductase (NTR) combined with systemic prodrug (CB1954) treatment. A single i.t. injection of CTL102 (7.5 x 10(9) to -2 x 10(10) particles) followed by CB1954 treatment produced clear anti-tumor effects in subcutaneous (s.c.) xenograft models of four cancers that are likely candidates for GDEPT (i.e., primary liver, head and neck, colorectal and prostate). Virus dose-response studies (s.c. liver model) revealed a steep increase and subsequent rapid plateauing of both NTR gene delivery and anti-tumor efficacy. Evidence of minor virus spread (toxicity) was observed in a s.c. head and neck xenograft model. This was eliminated by passive immunization with neutralizing anti-Ad5 antibodies prior to virus injection without reducing the magnitude of the anti-tumor effect. Preexisting anti-Ad5 neutralizing antibodies may therefore be an advantage rather than an issue in the clinical use of this new therapy.

Adenoviridae↗

PubMed2001

High-level, beta-catenin/TCF-dependent transgene expression in secondary colorectal cancer tissue.

There is an urgent need for improved therapies for inoperable metastatic colon cancer. Gene-directed enzyme prodrug therapy (GDEPT) using adenovirus vectors works well in preclinical models of this disease, but successful clinical application is hampered by an inability to construct vectors that express at high levels in infected tumor cells but not in infected normal cells. Constitutive activation of beta-catenin-dependent gene expression is almost certainly a key causative event in the genesis of colon and some other cancers. Here we have exploited this oncogenic defect to design a synthetic promoter, CTP1, that, in contrast to currently available tumor-selective promoters, is both highly active in cancer cells and highly cancer-cell-specific. CTP1 directs high-level beta-galactosidase expression in freshly isolated biopsies of secondary colon cancer, but is not detectably active in associated normal liver tissue. We also demonstrate that CTP1 can direct high-level, tumor-specific therapeutic gene expression in vivo. Intratumoral injection of an adenovirus vector encoding Escherichia coli nitroreductase driven by CTP1 efficiently sensitized SW480 xenografts to the prodrug CB1954, whereas systemic vector and prodrug administration produced no apparent signs of toxicity. CTP1 may form the basis for effective, targeted gene therapy of metastatic colon cancer and other tumors with deregulated beta-catenin/T cell factor.

Adenoviridae↗

PubMed1995

Distinct profile of working memory errors following acute or chronic disruption of the cholinergic septohippocampal pathway.

The behavioral effects of two amnestic treatments (intraseptal chlordiazepoxide (CDP) and intraventricular AF64A) were examined in a delayed-nonmatch-to-sample radial-arm maze (DNMTS) paradigm. The types of errors induced by these treatments in this working memory task were assessed to determine how acute and chronic disruptions of the medial septum affect different phases of working memory (encoding, maintenance, retrieval). Rats were initially trained to perform the DNMTS task with a 1-h delay imposed between the training and the testing sessions. The first experiment demonstrated that intraseptal injection of 30 nmoles of CDP did not produce state-dependent learning. Rats were injected immediately following training with CDP or artificial cerobrospinal fluid (CSF; drug vehicle) and then prior to testing with CDP or CSF. Injection of CDP immediately following training (CDP-CSF) impaired performance of the task regardless of whether CDP was also administered before the postdelay test (CDP-CDP). Rats infused with CDP only before the postdelay test (CSF-CDP) exhibited a proactive deficit characterized by intact retention of the predelay information (i.e., arms entered prior to the delay) but impaired performance on the postdelay component (arms entered only after the delay). These data indicate: (i) that state dependency does not explain the working memory deficits induced by intraseptal CDP; (ii) that pretest CDP disrupts the storage and utilization of working memory for current arm selections. The second experiment examined the behavioral effects induced by a permanent disruption of the cholinergic septohippocampal pathway produced by icv injection of the cholinotoxin AF64A. Rats were initially trained on the DNMTS task and then bilaterally injected icv with either AF64A (2.5 nmoles/side) or CSF. AF64A-treated rats exhibited a significant impairment of performance compared to CSF-treated controls. In contrast to the impairment exhibited by CDP-treated rats in Experiment 1, the performance of AF64A-treated rats displayed a deficit in the maintenance/retrieval of information acquired during RAM training and an impairment in ability to store current spatial information in working memory to guide postdelay testing performance. These studies demonstrate that acute and chronic disruptions of the septohippocampal pathway produce distinct profiles of cognitive impairment that should help to reveal the behavioral and neurobiological characteristics of spatial memory.

Animals↗

PubMed1997

Arecoline via miniosmotic pump improves AF64A-impaired radial maze performance in rats: a possible model of Alzheimer's disease.

Male Sprague-Dawley rats, preoperatively trained in a 1-h delay non-match-to-position radial maze task, received bilateral stereotaxic injections of a selective cholinotoxin, ethylcholine aziridinium ion (AF64A: 3 nmol/3 microliters/lateral ventricle). Animals treated with AF64A made significantly more total postdelay errors than vehicle controls. Sustained delivery, via miniosmotic pumps, of arecoline (0.1, 0.3, 1, 3, 10, or 30 mg/kg/day sc for 14 days) attenuated the AF64A-induced cognitive impairment in a dose-dependent manner, producing an inverted U-shaped dose-response function which was optimal at 1.0 mg/kg/day. Following these studies, choline acetyltransferase activity was significantly reduced in hippocampi extracted from the AF64A-treated rats, indicating successful cholinotoxicity. This paradigm may be useful as a possible screen for potential Alzheimer's disease therapeutic agents. This conclusion is supported by published reports of beneficial arecoline effects observed following 2-week intravenous infusions in patients with Alzheimer's disease (Soncrant, Raffaele, Asthana, Berardi, Morris, & Haxby, 1993).

Alzheimer Disease↗

PubMed1997

Evaluation of a live-attenuated foot-and-mouth disease virus as a vaccine candidate.

A variant of foot-and-mouth disease virus (FMDV) lacking the leader (L) coding region (A12-LLV2) was previously constructed and shown to be less virulent in cattle than its wild-type parent (A12-IC). In this study, cattle were tested for their clinical and immunological responses to subcutaneous inoculation with A12-LLV2 or A12-IC or to intramuscular vaccination with chemically inactivated A12-IC. Five weeks postinoculation animals were challenged by intradermal inoculation in the tongue with a virulent cattle-passaged virus. A12-LLV2-inoculated animals showed no clinical signs of disease and developed a neutralizing antibody response by 4 days postinoculation, whereas a companion control bovine did not seroconvert. After challenge, two of three inoculated animals did not develop lesions, but showed mild signs of infection. The third inoculated animal developed some lesions, but these were less severe than in the uninoculated control animal, which showed classical FMD. All animals inoculated with A12-IC developed a fever, two showed typical FMD lesions, and the companion control seroconverted, indicating that it had acquired infection by contact. The A12-IC-inoculated animals and the control were protected from challenge. Animals vaccinated with inactivated virus showed no clinical signs of disease and developed a neutralizing antibody response, and the control did not seroconvert. Upon challenge none of the vaccinated animals developed lesions, one developed a fever, and the control developed FMD. These experiments demonstrate the potential of a rationally designed live-attenuated FMDV vaccine.

Animals↗

PubMed1982