Phorbol esters rapidly attenuate glutamine uptake and growth in human colon carcinoma cells.

BACKGROUND: The amino acid glutamine, while essential for gut epithelial growth, has also been shown to stimulate colon carcinoma proliferation and diminish differentiation. Human colon carcinomas are known to extract and metabolize glutamine at rates severalfold greater than those of normal tissues, but the regulation of this response is unclear. Previously we reported that phorbol esters regulate hepatoma System ASC/B(0)-mediated glutamine uptake and cell growth. As human colon carcinoma cells use this same transporter for glutamine uptake, the present studies were undertaken to determine whether similar regulation functions in colon carcinoma. MATERIALS AND METHODS: Human colon carcinoma cell lines (WiDr and HT29) were treated with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and initial-rate transport of glutamine and other nutrients was measured at specific times thereafter. Growth rates were monitored during culture +/- PMA or an excess of System ASC/B(0) substrates relative to glutamine. RESULTS: PMA treatment induced a rapid inhibition of glutamine uptake rates in WiDr and HT29 cells by 30 and 57%, respectively, after 1 h. Cycloheximide failed to block this response, indicating that the mechanism by which PMA exerts its effects is posttranslational. The inhibition of glutamine uptake by PMA was abrogated by the PKC inhibitor staurosporine, suggesting that this rapid System ASC/B(0) regulation may be mediated by a PKC-dependent pathway. PMA also significantly decreased transport via System y(+) (arginine) and System A (small zwitterionic amino acids). Chronic phorbol ester treatment inhibited WiDr cell growth, as did attenuation of System B(0)-mediated glutamine uptake with other transporter substrates. CONCLUSIONS: System ASC/B(0) uptake governs glutamine-dependent growth in colon carcinoma cell lines, and is regulated by a phorbol ester-sensitive pathway that may involve PKC. The results further establish the link between glutamine uptake and colon carcinoma cell growth, a relationship worthy of further investigation with the goal of discovering novel cancer therapeutic targets.

Amino acid uptake and regulation in multicellular hepatoma spheroids.

BACKGROUND: Cancer cells maintained in monolayer tissue culture are frequently used to study tumor biology and nutrient uptake, but there is a concern that this system may not fully reflect clinical tumor physiology. Because cells grown in a 3-D configuration more closely resemble an in vivo environment, a model was developed and characterized for the growth of SK-Hep human hepatoma cells in suspension as multicellular tumor spheroids (MTS). The measurement of nutrient uptake in such a system has never been established. MATERIALS AND METHODS: SK-Hep cultures were initiated as single cell suspensions and grown as MTS in siliconized spinner flasks. The transport of several individual amino acids (arginine, glutamate, leucine, alpha-(N-methylamino)isobutyric acid (MeAIB), and glutamine (GLN)) was measured in SK-Hep single cell suspensions and MTS (0. 50-0.60 mm diameter) by a radiotracer/rapid filtration technique, as was the regulation of glutamine uptake by phorbol esters. l-[(3)H]GLN uptake was also measured in larger spheroids (0.85-1.5 mm diameter). MTS cellularity was evaluated by histological examination, and single cell integrity after the transport assay was confirmed by scanning electron microscopy (SEM). RESULTS: SK-Hep MTS displayed gradients of cellular morphology and staining, with central necrosis visible at diameters >0.8 mm. Single cell suspensions endured the rapid filtration technique based on functional Na(+)-dependent uptake rates and SEM analysis. Of all amino acids tested, only GLN transport rates were visibly affected by growth format. In small MTS, Na(+)-dependent GLN uptake was diminished by 40%, but was 40-53% higher in MTS >1 mm displaying central necrosis, when compared to single cell suspensions. Likewise, slight parallel changes in glutamine transporter ATB(0) mRNA levels were observed in Northern blot analysis. Finally, phorbol ester-dependent GLN transport down-regulation (by 40-50%), previously established in SK-Hep monolayers, remained operative in all cell formats tested. CONCLUSIONS: The data suggest that the tumor microenvironment differentially impacts the uptake of specific nutrients despite the conservation of key regulatory pathways. This MTS technique may prove useful for further studies on the role of nutrient transport in nascent tumor growth.

Visualizing the kinetics of tumor-cell clearance in living animals.

Evaluation of potential antineoplastic therapies would be enhanced by noninvasive detection of tumor cells in living animals. Because light is transmitted through mammalian tissues, it was possible to use bioluminescence to monitor (both externally and quantitatively) growth and regression of labeled human cervical carcinoma (HeLa) cells engrafted into immunodeficient mice. The efficacy of both chemotherapy and immunotherapeutic treatment with ex vivo expanded human T cell-derived effector cells was evaluated. In the absence of therapy, animals showed progressive increases in signal intensity over time. Animals treated with cisplatin had marked reductions in tumor signal; 5'-fluorouracil was less effective, and cyclophosphamide was ineffective. Immunotherapy dramatically reduced signals at high effector-to-target cell ratios, and significant decreases were observed with lower ratios. This model system allowed sensitive, quantitative, real-time spatiotemporal analyses of the dynamics of neoplastic cell growth and facilitated rapid optimization of effective treatment regimens.

Noninvasive assessment of tumor cell proliferation in animal models.

Revealing the mechanisms of neoplastic disease and enhancing our ability to intervene in these processes requires an increased understanding of cellular and molecular changes as they occur in intact living animal models. We have begun to address these needs by developing a method of labeling tumor cells through constitutive expression of an optical reporter gene, and noninvasively monitoring cellular proliferation in vivo using a sensitive photon detection system. A stable line of HeLa cells that expressed a modified firefly luciferase gene was generated, and proliferation of these cells in irradiated severe combined immunodeficiency (SCID) mice was monitored. Tumor cells were introduced into animals via subcutaneous, intraperitoneal and intravenous inoculation and whole body images, that revealed tumor location and growth kinetics, were obtained. The number of photons that were emitted from the labeled tumor cells and transmitted through murine tissues was sufficient to detect 1x10(3) cells in the peritoneal cavity, 1x10(4) cells at subcutaneous sites and 1x10(6) circulating cells immediately following injection. The kinetics of cell proliferation, as measured by photon emission, was exponential in the peritoneal cavity and at subcutaneous sites. Intravenous inoculation resulted in detectable colonies of tumor cells in animals receiving more than 1x10(6) cells. Our demonstrated ability to detect small numbers of tumor cells in living animals noninvasively suggests that therapies designed to treat minimal disease states, as occur early in the disease course and after elimination of the tumor mass, may be monitored using this approach. Moreover, it may be possible to monitor micrometastases and evaluate the molecular steps in the metastatic process. Spatiotemporal analyses of neoplasia will improve the predictability of animal models of human disease as study groups can be followed over time, and this method will accelerate development of novel therapeutic strategies.

Early, aggressive therapy for rheumatoid arthritis: concerns, descriptions, and estimate of outcome.

The past few years have witnessed changing perceptions about rheumatoid arthritis (RA); it is now considered a serious systemic disease that confers not only physical and social morbidity but also earlier mortality. The long-term outcome of sequential monotherapy based on the therapeutic pyramid has been disappointing. A review of prognostic factors, acute disease activity measures, functional measures, and the results of preliminary trials with combination therapy suggests that specific goals of treatment can be established and that logical, aggressive treatment in early disease can be accomplished. These goals should include prompt control and continuous reduction of the active joint count to < or = 4 and normalization of acute-phase reactants. The "graduated-step paradigm" of treatment designed with these goals in mind is described, and a retrospective series that gives an estimate of outcome with its use is reported.

Deployment problems with the titanium Greenfield filter.

PURPOSE: The authors retrospectively reviewed their initial experience with deployment of the modified hook titanium Greenfield filter. PATIENTS AND METHODS: Twenty-three patients underwent filter placements over a 1-year period. Radiographs were obtained immediately after placement to confirm filter position in all cases. Follow-up images were available in 15 patients (65%). RESULTS: Twenty-four filters were placed in 23 patients. Tilting of the filter (> 15 degrees) was evaluated in 22 placements without complications and was present in five (23%). In 17 of 24 placements (71%), distribution of filter legs was poor, with wide gaps between clustered legs. Manipulation of the filter legs with an angiographic catheter resulted in improved distribution in three of six attempts but also resulted in a caudal displacement, which necessitated placement of a second filter. At follow-up (range, 4-16 months; mean, 9 months), three cases of asymptomatic inferior vena caval thrombosis and one recurrent pulmonary embolism were discovered. CONCLUSION: No untoward event resulting from filter placement was demonstrated. Further study and review of the deployment mechanism may be necessary.