Ascorbic acid and cell survival of adriamycin resistant and sensitive MCF-7 breast tumor cells.

The ability of human cells to regenerate ascorbic acid from dehydroascorbate is partially dependent on the glutathione redox status of the cell and the relative activity of dehydroascorbate reductases. Mammalian dehydroascorbate reductase activity is associated with two proteins known as thioltransferase (glutaredoxin) and protein disulfide isomerase. We compared the specific activity of thioltransferase, protein disulfide isomerase, and other GSH-related enzymes in Adriamycin-resistant human breast tumor cells, MCF-7 ADRR, and Adriamycin-sensitive, MCF-7 WT, tumor cells. MCF-7 ADRR cells had higher activities of glutathione peroxidase (34.7 fold), nonseleno-glutathione peroxidase (glutathione S-transferases; 5.3 fold), thioredoxin (2.3 fold), and thioltransferase (4.0 fold) compared with the WT Adriamycin-sensitive cell line. Thioltransferase was detected in Western blots in extracts of ADRR MCF-7 cells but not in WT MCF-7 cells. alpha-Tocopherol in the membrane and cytosolic fractions was 2.8 and 3.0 fold higher, respectively, in Adriamycin-resistant compared with Adriamycin-sensitive cells. Supplementation of MCF-7 cells with L-ascorbic acid 2-phosphate (2 and 10 mM) had no effect on WT cell viability after 5 days incubation with up to 0.33 microM Adriamycin. In contrast, supplementation of ADRR MCF-7 cells with L-ascorbic acid 2-phosphate resulted in enhanced resistance up to 3.4 microM Adriamycin over a 5-day incubation. Both lines of MCF-7 cells demonstrated the ability to utilize ascorbic acid as the 2-phosphate derivative. After 48 h incubation with 8.6 microM Adriamycin, the resistant cells maintained normal viability and ascorbate-dehydroascorbate levels, whereas drug-sensitive cells had significantly lower ascorbate with a higher percent dehydroascorbate and increased cell death as judged by cell protein levels (52% of controls).

Immunoscintigraphy with a new indium-111-labeled monoclonal antibody (MAb 1A3) in patients with colorectal cancer.

PURPOSE: This study was designed to evaluate a new anticolorectal carcinoma monoclonal antibody (1A3), conjugated with the bifunctional chelating agent N,N'-bis(2-hydroxybenzyl)1(4-bromoacetamidobenzyl)1,2-ethylenediam ine-N,N'- diacetic acid and labeled with indium-111, in a Phase I/II study involving 38 patients with localized or advanced colorectal cancer. METHODS: Patients were injected with indium-111-N,N'-bis(2-hydroxybenzyl) 1(4-bromoacetamidobenzyl)1,2-ethylenediamine-N,N'-diacetic acid-monoclonal antibody 1A3 (1-50 mg, 1-5 mCi) and imaged at two or three sessions one to five days later. Scintigraphic findings were compared with radiologic, pathologic, surgical, and other clinical findings to assess the accuracy of radioimmunoscintigraphy. RESULTS: At least one known tumor site was clearly defined by planar scintigraphy in 29 (76 percent) patients. Increased radioactivity was seen in 40 of 63 known tumor sites (37/43 abdominal-pelvic, 3/15 hepatic, and 0/5 pulmonary sites) without any apparent dose-related effects. Nineteen previously undetected sites were considered positive by imaging, and, of these, six were biopsy-proven tumor sites, four were probable tumor sites, three were definitely false positive sites, and six were probable false positive sites. Radioimmunoscintigraphy detected proven tumor in 15 of 16 patients with negative or equivocal computed tomography results. Of of the 28 patients with rectosigmoid cancer, 25 (89 percent) had positive studies with 34 of 47 tumor sites showing definite uptake on the scintigrams. This included 3 of 9 hepatic metastases. The only adverse reaction occurred in one patient who developed transient hives. Human anti-mouse antibody responses occurred in approximately one-half of the patients injected with doses of 10 or 50 mg. CONCLUSION: This study shows that radioimmunoscintigraphy with this indium-111-labeled monoclonal antibody is safe, it can detect most nonhepatic abdominal-pelvic tumors with a positive predictive value of 83 (44/53) percent, and it should prove to be useful, particularly in the diagnosis of recurrent rectal carcinoma.

Evaluation of a direct method for technetium labeling intact and F(ab')2 1A3, an anticolorectal monoclonal antibody.

A direct method for 99mTc-labeling monoclonal antibodies (MAb) has been evaluated for labeling intact and F(ab')2 1A3, an anticolorectal carcinoma MAb. The method employs ascorbic acid to reduce the MAbs. By altering the reaction conditions for 99mTc-1A3, a maximum radiolabeling yield of 48% was obtained with an immunoreactivity (IR) value of 87%; and for 99mTc-1A3-F(ab')2, a yield of 49% and an IR value of 70% was obtained. Biodistribution of 99mTc-labeled 1A3 MAbs was performed in a Golden Syrian hamster model and compared to 125I-labeled 1A3 MAbs. Tumor uptake (%ID/g) was significantly better for the intact 125I-1A3 at 24 h post-injection compared to the intact 99mTc-1A3. For 99mTc-1A3-F(ab')2, %ID/g tumor was low, and did not increase over 24 h. High %ID/g kidney persisted at 24 h for both 99mTc-labeled intact and F(ab')2 1A3. Serum stability was performed in Sprague-Dawley rats for the 99mTc-labeled 1A3 MAbs, and compared to 125I-labeled 1A3 MAbs, which showed intact 99mTc-1A3 cleared similarly to 125I-1A3, and 99mTc-1A3-F(ab')2 cleared more rapidly than 125I-1A3-F(ab')2 indicating instability of the 99mTc-labeled 1A3-F(ab')2.

Evaluation of copper-labeled bifunctional chelate-albumin conjugates for blood pool imaging.

62Cu(T1/2 = 9.8 min) is a generator-produced positron-emitting radionuclide with a half-life amenable to blood-pool imaging with PET. Three bifunctional chelates [cyclic anhydride of diethylenetriaminepentaacetic acid (cDTPAA), 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N ',N", N"'-tetraacetic acid (BAT), and p-carboxyethylphenylglyoxal-bis-(4N-methyl-thiosemicarbazone (CE-DTS)] were conjugated to HSA and labeled with 67Cu. The labeling efficiency of 67Cu-DTS-HSA was > 90%, whereas the labeling yields of 67Cu-DTPA-HSA and 67Cu-benzyl-TETA-HSA were less than 70%. Blood clearance and biodistribution of these three 67Cu-labeled conjugates were determined in rats. Of the three 67Cu-labeled bifunctional chelate-HSA conjugates, 67Cu-benzyl-TETA-HSA remained in the blood pool the longest, achieving stable blood levels at times longer than 24 h post-injection. The 67Cu radioactivity cleared the blood within 60 min post-injection of 67Cu-DTS-HSA, and within 10 min after administration of 67Cu-DTPA-HSA, indicating the dissociation of Cu2+ from these conjugates. Copper-labeled DTS-HSA achieved stable blood concentrations for at least 30 min post-injection and was therefore evaluated as a vascular imaging agent. DTS-HSA and benzyl-TETA-HSA were labeled with 62Cu and administered to a dog for blood-pool imaging using PET. Images were nearly identical to an image taken after administration of C15O. Because of the high labeling efficiency, DTS-HSA can be labeled with 62Cu without purification, making it more practical than 62Cu-benzyl-TETA-HSA as a blood-pool imaging agent.(ABSTRACT TRUNCATED AT 250 WORDS)

16 beta-[18F]fluoromoxestrol: a potent, metabolically stable positron emission tomography imaging agent for estrogen receptor positive human breast tumors.

16 beta-[18F]Fluoromoxestrol (beta FMOX, 1) is a highly selective, metabolically stable estrogen with potential as a receptor imaging agent. It demonstrates receptor-mediated uptake in the immature rat in the estrogen receptor-rich primary target tissues, uterus and ovaries, as well as, in receptor-poor secondary target tissues, muscle, thymus and kidneys; uptake in the uterus is nearly four times that of the clinically useful 16 alpha-[18F]fluoroestradiol (FES), most likely due to the extended lifetime of the labeled beta FMOX in the blood afforded by its relatively slow metabolism. In vivo and in vitro studies demonstrate a nearly four-fold decrease in metabolism rate between beta FMOX and FES. Dosimetry studies indicate radiation absorbed doses comparable to FES. beta FMOX possesses desirable imaging characteristics and may prove to be a clinically useful imaging agent.

Copper-64-labeled antibodies for PET imaging.

In the imaging of tumors using radiolabeled monoclonal antibodies, the use of PET gives increased sensitivity over conventional gamma camera imaging techniques. Copper-64, a positron-emitting radionuclide, has been labeled to 1A3, an anticolorectal carcinoma monoclonal antibody, and its fragments 1A3-F(ab')2 utilizing the bifunctional chelate Br-benzyl-TETA. The 64Cu-labeled intact 1A3 and 1A3-F(ab')2 have been evaluated as potential imaging agents for PET. Biodistribution studies of 64Cu-benzyl-TETA-1A3 and 64Cu-benzyl-TETA-1A3-F(ab')2 in tumor-bearing hamsters were compared with those of 111In-Br phi HBED-1A3, 111In-Br phi HBED-1A3-F(ab')2 and 125I-labeled intact 1A3 and 1A3-F(ab')2. Tumor uptake of 64Cu-labeled intact 1A3 and fragments in the hamster model was superior to both 111In- and 125I-labeled intact 1A3 and fragments. Human dosimetry data for 64Cu- and 123I-labeled 1A3 and 1A3-F(ab')2 were calculated from biodistribution data in rats. High kidney uptake of 64Cu-benzyl-TETA-1A3-F(ab')2 precludes clinical study at this time; however, the data shows that 64Cu-benzyl-TETA-1A3 would be suitable for positron tomography imaging of colorectal cancer in patients.

Interactions of platinum complexes with thioltransferase(glutaredoxin), in vitro.

Under anaerobic conditions, recombinant pig liver thioltransferase (glutaredoxin)(TT, GRX) (EC 1.8.4.1) was strongly inhibited by cis and carbo-platin and somewhat less sensitive to trans-platin, in vitro. By extrapolation to total inhibition, the ratio of platinum drug/thioltransferase was approximately 1.0 for cis and carbo-platin, but greater than 1.0 for trans-platin. When thioltransferase was not reduced, inhibition by preincubation with the platinum complexes required molar excesses of 1,300 and 675 to one for cis-platin and trans-platin, respectively or 400-500 microM for 50% inhibition. The inhibition of thioltransferase at high drug concentrations in the presence of oxygen was associated with cross-linking of monomers into dimers within 5 min and, in the case of cis-platin treatment, to trimers in 20 min incubation.

Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity.

Homogeneous native and recombinant porcine liver thioltransferase (glutaredoxin), bovine thymus and human placenta thioltransferase (glutaredoxin) were examined for dehydroascorbate reductase activity (EC 1.8.5.1) involving the direct catalytic reduction of dehydroascorbic acid (DHA) by glutathione. Each enzyme had substantial activity with apparent Km and Vmax for dehydroascorbate between 0.2 and 2.2 mM and 6-27 nmol min-1, respectively, and for gluathione between 1.6 and 8.7 mM and 11-30 nmol min-1, respectively. In the presence of purified bovine liver thioredoxin reductase, homogeneous bovine liver thioredoxin failed to reduce DHA to ascorbic acid as measured by NADPH oxidation. Highly purified bovine liver protein disulfide isomerase (PDI) reacted directly with DHA and GSH to catalyze the reduction of DHA to ascorbic acid. The apparent Km for DHA was 1.0 mM and the Vmax was 8 nmol min-1, and for GSH were 3.9 mM and 14 nmol min-1, respectively. These results suggest that thioltransferase and PDI contribute to the regeneration of oxidized ascorbic acid in mammalian cells, and based on their cellular location, thioltransferase is proposed to be the major cytoplasmic activity, whereas interaction of DHA with microsomal membrane PDI may catalyze regeneration of ascorbic acid and initiate oxidation of intralumenal protein thiols to disulfides.