Quantitative 19F NMR study of trifluorothymidine metabolism in rat brain.

Metabolism of trifluorothymidine (TFT) and its transport across the blood-brain barrier (BBB) has been measured quantitatively in rats by fluorine-19 nuclear magnetic resonance spectroscopy ((19)F NMR). It is demonstrated that TFT crosses the BBB in micromolar quantities and is metabolized in brain tissue primarily to its free base trifluoromethyluracil (TFMU) by the enzyme thymidine phosphorylase (TP). It is further proposed that the rate of TFMU production can be used as a measure of cerebral TP. The glycols of both TFMU, and to a lesser degree TFT, are generated via an oxidative route. In contrast, the major pathway for hepatic metabolism of this compound is through reduction of the nitrogen base moiety and generation of 5-6-dihydro species followed by ring degradation. Thus, in addition to TFMU as well as the dihydroxy (glycol)-, and the dihydro-species of both TFT and TFMU, alpha-trifluoromethyl-beta-ureidopropionic acid (F(3)MUPA) and alpha-trifluoromethyl-beta-alanine (F(3)MBA) were detected in liver extracts. The total metabolite levels in liver were 2-5 times higher than in the brain. Low levels of fluoride ion were detected in all the extracts from brain and liver, as well as blood and urine. This study characterizes TFT as a potential chemotherapeutic agent for use against brain tumors.

Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO(2) or pH.

Accumulation of CO(2) in animal cell cultures can be a significant problem during scale-up and production of recombinant glycoprotein biopharmaceuticals. By examining the cell-surface polysialic acid (PSA) content, we show that elevated CO(2) partial pressure (pCO(2)) can alter protein glycosylation. PSA is a high-molecular-weight polymer attached to several complex N-linked oligosaccharides on the neural cell adhesion molecule (NCAM), so that small changes in either core glycosylation or in polysialylation are amplified and easily measured. Flow-cytometric analysis revealed that PSA levels on Chinese hamster ovary (CHO) cells decrease with increasing pCO(2) in a dose-dependent manner, independent of any change in NCAM content. The results are highly pH-dependent, with a greater decrease in PSA at higher pH. By manipulating medium pH and pCO(2), we showed that decreases in PSA correlate well with bicarbonate concentration ([HCO(3)(-)]). In fact, it was possible to offset a 60% decrease in PSA content at 120 mm Hg pCO(2) by decreasing the pH from 7.3 to 6.9, such that [HCO(3)(-)] was lowered to that of control (38 mm Hg pCO(2)). When the increase in osmolality associated with elevated [HCO(3)(-)] was offset by decreasing the basal medium [NaCl], elevated [HCO(3)(-)] still caused a decrease in PSA, although less extensive than without osmolality control. By increasing [NaCl], we show that hyperosmolality alone decreases PSA content, but to a lesser extent than for the same osmolality increase due to elevated [NaHCO(3)]. In conclusion, we demonstrate the importance of pH and pCO(2) interactions, and show that [HCO(3)(-)] and osmolality can account for the observed changes in PSA content over a wide range of pH and pCO(2) values.

Role of nucleotide sugar pools in the inhibition of NCAM polysialylation by ammonia.

Ammonia in animal cell cultures has been shown to specifically inhibit terminal sialylation of N- and O-linked oligosaccharides of glycoproteins. For example, we have previously shown that as little as 2.5 mM NH4Cl can decrease neural cell adhesion molecule (NCAM) polysialylation in both small cell lung cancer (SCLC) and Chinese hamster ovary (CHO) cells. Besides its potential involvement in SCLC metastasis, polysialic acid (PolySia) is a sensitive marker for measuring changes in sialylation. The role of UDP-N-acetylglucosamine (UDP-GlcNAc) in ammonia's inhibition of NCAM polysialylation was examined by adding glucosamine (GlcN) and uridine (Urd) to the cultures. This bypassed feedback inhibition of GlcN-6-P synthase and increased UDP-GlcNAc content by 25-fold in SCLC cells. After 3 days, PolySia levels were reduced to 10% of control with little effect on NCAM protein content. The extensive decrease in PolySia was confirmed in CHO cells. The effects of GlcN or Urd alone were less extensive, lending support to a specific role for UDP-GlcNAc in inhibition by ammonia. By comparison, 20 mM NH4Cl decreased PolySia content by 45% and increased UDP-GlcNAc in SCLC cells by 2-fold. The discrepancy between the ¿GlcN+Urd¿ and NH4Cl effects on UDP-GlcNAc and PolySia suggests that accumulation of UDP-GlcNAc is only partially responsible for decreased polysialylation in response to NH4Cl. In an attempt to increase NCAM polysialylation, N-acetylmannosamine and cytidine were added to cultures in order to circumvent the feedback inhibition of CMP-sialic acid synthesis. However, this only slightly increased PolySia levels and failed to counter ammonia's inhibition of NCAM polysialylation.

Ammonia inhibits neural cell adhesion molecule polysialylation in Chinese hamster ovary and small cell lung cancer cells.

Ammonia is a major concern in biotechnology because it often limits recombinant protein production by animal cells. Conditions, such as ammonia accumulation, in large-scale production systems can parallel those that develop within fast-growing solid tumors such as small cell lung cancer (SCLC). Ammonia's specific inhibition of the sialylation of secreted glycoproteins is well documented, but it is not known how ammonia affects membrane-bound proteins, nor what role it may have on important glycosylation determinants in cancer. We therefore examined the effects of NH4Cl on polysialic acid (PolySia) in the neural cell adhesion molecule (NCAM). By using flow cytometry combined with two NCAM antibodies, one specific for the peptide backbone and another that recognizes PolySia chains, we show that ammonia causes rapid, dose-dependent, and reversible inhibition of NCAM polysialylation in Chinese hamster ovary (CHO) and SCLC NCI-N417 cells. The decrease in PolySia was accompanied by a small increase in NCAM, suggesting that the changes were specific to the oligosaccharide. Inhibition by ammonia was greater for CHO cells, with PolySia cell surface content decreasing to 10% of control after a 4-day culture with 10 mM NH4Cl, while N417 cell PolySia was reduced by only 35%. Ammonia caused a 60% decrease in the CHO cell yield from glucose, while N417 cells were barely affected, suggesting that increased resistance to ammonia by N41 7 cells is a global rather than glycosylation-specific phenomenon. The data presented show that the tumor microenvironment may be an important factor in the regulation of PolySia expression.

Discrimination of fluorinated uridine metabolites in N-417 small cell lung cancer cell extracts via 19F- and 31P-NMR.

31P-NMR extract spectra of N-417 Small Cell Lung Cancer (SCLC) cells cultured with fluorouridine (FUrd) reveal new peaks with chemical shifts in the diphosphodiester and nucleoside triphosphate regions. These peaks were identified as FUTP, FUDP, FUDP-glucose, FUDP-glucuronate, FUDP-GlcNAc, and FUDP-GalNAc via enzymatic conversion and 19F- and 31P-NMR analysis. Distinct 19F chemical shifts were assigned for FUTP, FUDP, and the FUDP-sugars.

High-dose aminothiadiazole in advanced colorectal cancer. An Illinois Cancer Center phase II trial.

Thirty-three patients with advanced colorectal carcinoma were entered on a phase II trial of weekly IV aminothiadiazole (175 mg/m2 escalated to 200 mg/m2) with concomitant allopurinol and non-steroidal anti-inflammatory agents (NSAID's). Toxicity was predominantly GI, cutaneous, and chest pain/dyspnea. Twenty-five percent of patients had grade 3 or 4 toxicity. There were no responses in 27 evaluable patients. Median survival was 12 months. Aminothiadiazole, at higher doses than used in previous reports, when given with NSAID's, had no significant activity against large bowel cancer.

Detecting fatty acids of dietary origin in normal and cancerous human breast tissue by 13C nuclear magnetic resonance spectroscopy.

Natural abundance 13C NMR was used to determine relative amounts of fatty acid subclasses present in fibroadipose tissue from the human breast in healthy and cancer patients and in breast carcinoma tissue. Resonances corresponding to the carbon atoms of triacylglycerides were obtained when adipose tissue constituted more than 10% of the carcinoma. Resonances corresponding to phospholipids and proteins were also observed when the percentage of adipose tissue was lower. No significant difference between the levels of unsaturated fatty acids in adipose tissue from cancer and non-cancer patients was found. However, significant differences in the levels of monounsaturated and saturated fatty acids of carcinoma compared to non-cancerous tissue was found, as was a nearly significant difference for the levels of polyunsaturated fatty acids in these two tissue types. These findings suggest an alteration of cellular lipid composition in neoplastic mammary tissue.

UDP-N-acetylhexosamine modulation by glucosamine and uridine in NCI N-417 variant small cell lung cancer cells: 31P nuclear magnetic resonance results.

Small cell lung cancer (SCLC) occurs as two neuroendocrine subtypes, SCLC-C (classic) and SCLC-V (variant). One reported difference is elevated levels of diphosphodiesters (DPDE) in the more differentiated SCLC-C subtype. DPDE have been identified as primarily UDP-N-acetylhexosamines (UDP-NAH) in a variety of tumors, and changes in DPDE levels have been observed during experiments designed to induce cell differentiation. UDP-NAH synthesis is controlled by negative feedback regulation of glutamine:fructose-6-P amidotransferase (EC 2.6.1.16), which can be circumvented by glucosamine. Using 31P nuclear magnetic resonance analysis of extracts and perfused cells, we have identified UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine as the primary metabolites in the DPDE spectral region of SCLC-V N-417 cells. Glucosamine addition causes a rapid increase in UDP-NAH levels. At glucosamine: glucose ratios of 1:1 and 10:1 formation of the UDP-NAH intermediates N-acetylglucosamine 6-phosphate and UDP-N-acetylglucosamine 1-phosphate is also observed, indicating UTP limitation. Subsequent uridine addition results in depletion of the intermediates and increased UDP-NAH formation. Moreover, N-417 cells retain the capacity to rapidly convert uridine to UTP despite low ATP and phosphocreatine levels. This expansion of the uridine pool may represent an additional metabolic reserve not yet addressed in the design of therapy options.

A reproducible model of metastatic brain and ocular tumor by hematogenous inoculation of the VX2 tumor in rabbits.

A metastatic brain-tumor model has been developed in rabbits by infusing the VX2 carcinoma into the internal carotid artery to simulate hematogenous dissemination of tumor. In a series of 25 New Zealand White rabbits, multiple metastases arose in the hemisphere of 24 (96%) and in the eye of 22 (92%); in all instances ocular metastases were ipsilateral to the site of infusion. Ocular metastases were visible in the anterior chamber in 80% of animals 3 to 12 days after the infusion of VX2 tumor cell suspension. All rabbits deteriorated neurologically or died by Day 15 after the inoculation. Multiple metastases were demonstrated by magnetic resonance imaging as early as 5 to 7 days after infusion of the tumor cells and were confirmed at autopsy. This technique models hematogenous metastases to the brain and eye and is useful in evaluating the response of metastases to chemotherapy and radiation therapy directed to the brain and eye.

Levels of high energy phosphates in human lung cancer cell lines by 31P nuclear magnetic resonance spectroscopy.

Human lung cancers are divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) based on established criteria. SCLC differs from NSCLC by the expression of biomarkers, including creatine kinase-BB isoenzyme (EC 2.7.3.2). Subtypes of SCLC are referred to as classic and variant, both of which have elevated levels of creatine kinase-BB isoenzyme. We, therefore, applied 31P nuclear magnetic resonance spectroscopy to cell lines of classic SCLC, variant SCLC, and NSCLC human tumors, using continuous perfusion to identify any differences in the detectable levels of intracellular high-energy phosphate compounds. The spectra indicate that only the variant SCLC cells maintain high levels of phosphocreatine. Additionally, the classic SCLC cells express elevated levels of a diphosphodiester. Neither phosphocreatine nor diphosphodiesters are found in the NSCLC cell spectra.

Magnetic resonance imaging versus computed tomography in the evaluation of soft tissue tumors of the extremities.

Twenty patients with extremity soft tissue tumors were prospectively evaluated with magnetic resonance imaging (MRI) and computed tomography (CT) scans with subsequent anatomic correlation of surgical findings. MRI and CT had a similar percentage of accuracy in assessing tumor relationship with major neurovascular (80% and 70%, respectively) and skeletal (80% and 75%, respectively) structures. MRI was significantly better than CT in displaying contrast between tumor and muscle when using the T2 weighted spin echo (SE) (p2 less than 0.002) and inversion recovery (IR) (p2 less than 0.005) pulse sequences. MRI and CT were comparable in demonstrating contrast between tumor and fat. The contrast between tumor and vessel was better displayed by MRI compared with CT when using the T1 weighted SE (p2 less than 0.001) and T2 weighted SE (p2 less than 0.001) pulse sequences. T1 and T2 values were measured on fresh tumor and normal tissue samples and were used to predict relative contrast on different MRI pulse sequences using isosignal contour plots. MRI appears to offer several advantages over CT in the evaluation of extremity soft tissue tumors.

Gadolinium cryptelates as MR contrast agents.

Gadolinium cryptelates are complexes of a lanthanide metal ion with amino acids of macrocyclic polyamines. These compounds are water soluble and possess reduced relaxation properties similar to Gd diethylene triamine pentaacetic acid (DTPA). Three Gd cryptelates (Gd NOTA, DOTA, TETA) were evaluated. Gadolinium DOTA is the most stable Gd complex with a dissociation constant of 10(-28) and appears to have a greater serum stability than Gd DTPA. Gadolinium NOTA and Gd TETA have lower dissociation constants than Gd DTPA at 10(-17) and 10(-19). Gadolinium DOTA has tissue distribution properties similar to Gd DTPA, is rapidly excreted by the kidneys, and provides a high degree of contrast enhancement on magnetic resonance (MR) images, both systemically and within the CNS. Hence, Gd DOTA is an alternative water-soluble MR contrast agent to Gd DTPA.

Opening of blood-ocular barrier demonstrated by contrast-enhanced MR imaging.

Opening of the blood-ocular barrier following infusion of hyperosmolar agents into the internal carotid artery has been demonstrated by gadolinium enhanced magnetic resonance (MR) imaging. In five rhesus monkeys the disruption of the barrier was shown as increased signal intensity within the aqueous and vitreous humors. These findings suggest a potential use of contrast-enhanced MR imaging for detecting and evaluating the ocular microangiopathy of diabetic and hypertensive retinopathy and other diseases.

Gd(DOTA): an alternative to Gd(DTPA) as a T1,2 relaxation agent for NMR imaging or spectroscopy.

Methods have been devised for obtaining gadolinium(III) complexes of the ligands NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), and DTPA (diethylenetriaminepentaacetic acid) as solids for use as pharmaceuticals. Their effectiveness as in vitro and in vivo contrast agents for NMR imaging or T1,2 relaxation agents for spectroscopy has been investigated. The Gd(DOTA) complex was shown to be a more stable alternative to Gd(DTPA) by serum stability studies and measurement of stability constants. Images of tumors grown in athymic mice were obtained by NMR after injection of Gd(DOTA) and Gd(DTPA).

Metalloporphyrin contrast agents for magnetic resonance imaging of human tumors in mice.

Enhanced contrast of transplanted human colon carcinoma in athymic mice is seen in magnetic resonance images upon iv injection of the complexes of Mn(III) with two water-soluble porphyrins, tetra (4-sulfonatophenyl) porphyrin and tetra (N-methyl-4-pyridyl) porphyrin.

Adrenal masses differentiated by MR.

Magnetic resonance imaging was performed in 30 patients with adrenal masses. The abnormalities included adrenal adenomas (n = 10), carcinomas (n = 2), pheochromocytomas (n = 12), and adrenal metastases (n = 6). By the ratio of the signal intensity of the adrenal mass to that of the liver, adenomas could be distinguished from adrenal metastases, adrenal carcinomas, and pheochromocytomas. Metastases and pheochromocytomas could generally be differentiated.

MR cisternography and myelography with Gd-DTPA in monkeys.

To enhance the contrast between cerebrospinal fluid (CSF), brain, spinal cord, and surrounding meninges and bone on magnetic resonance (MR) images, as well as to study CSF flow, gadolinium-DTPA was injected in the subarachnoid space of eight monkeys. Six doses of progressively higher concentrations (from .125 mmol to 250 mmol) were injected every 30-40 minutes. Images of head and spine were obtained at .26 T or .5 T in sagittal and axial planes, using both spin-echo and inversion-recovery sequences in 13 imaging experiments. Marked, consistent changes of signal intensity in the CSF cavities were observed following the injections. These changes were dose related and occurred at different times in the areas close to the injection site versus those distant, a disparity that obviously was related to CSF flow. Gd-DTPA cisternography and myelography may be valuable in MR imaging of central nervous system disease, such as tumors adjacent to the CSF cavities, abnormal CSF collections (e.g., arachnoidal cysts), CSF rhinorrhea and otorrhea, syringohydromyelia, and studies of hydrocephalus and CSF flow dynamics.

Distinction between adrenal adenomas and metastases using MR imaging.

Magnetic resonance imaging was performed on 12 patients with known neoplastic disease and adrenal masses shown by CT. Three patients with metastases had high signal intensity in the adrenals on T2 weighted spin echo scans (SE 2,500/80) and nine patients with nonfunctioning adenomas had low signal intensity on T2 weighted images. The ability to distinguish metastases from nonhyperfunctioning adrenal adenomas may be of use in the pre-operative evaluation in patients with known carcinoma and incidental adrenal masses.

Tumors and arteriovenous malformations of the spinal cord: assessment using MR.

Thirty-three patients with either primary spinal cord tumors (n = 18), intradural tumors excavating into the cord (n = 9), or spinal arteriovenous malformations (AVM) (n = 6) were studied with magnetic resonance (MR) imaging. In 25 of 38 examinations (66%) (five patients were studied twice), MR provided more information than that provided by other neuroradiologic procedures. In several cases, MR affected patient management decisions. Advantages of MR, in addition to the absence of ionizing radiation and its noninvasive nature, include good spinal cord-CSF-theca contrast, lack of bone-derived artifacts, ease of multiplanar imaging, improved discrimination between intra- and extramedullary lesions, better definition of tumoral cavities and possible distinction from true syringes, ability to help one recognize thrombus formation within an AVM, and ease of follow-up of cord lesions for possible size changes. Apart from factors precluding the study in several patients (life support systems, pacemakers, claustrophobia, neurovascular clips), disadvantages of MR imaging include motion artifacts (prevalent in thoracolumbar area), poor capability of typing and grading of tumors, potential of false-positive results, poor detection of calcification, and poor delineation of feeders and drainers of AVM.