Cyclocreatine accumulation leads to cellular swelling in C6 glioma multicellular spheroids: diffusion and one-dimensional chemical shift nuclear magnetic resonance microscopy.

Cyclocreatine, an analogue of creatine, inhibits tumor cell proliferation in vitro and in vivo. The effects of cyclocreatine in large C6 glioma multicellular spheroids were mapped here by magnetic resonance microscopy. Diffusion-weighted images of C6 glioma spheroids resolved the bright viable rim and the dark necrotic center. Sequential sets of diffusion images, following cyclocreatine administration, showed increasing self-diffusion coefficients of the intracellular water in the viable rim (0.49 x 10(-5) cm2/s for untreated spheroids, 0.62 x 10(-5) cm2/s after 48 h perfusion with 20 mM cyclocreatine). This fact correlated with cellular swelling apparent in histological sections. The radial distribution of cyclocreatine and soluble lipids across perfused C6 spheroids was measured by one-dimensional chemical shift imaging. Cyclocreatine accumulation was prominent throughout the viable cell layer, with no cyclocreatine accumulation in the necrotic center. In both cyclocreatine-treated and control spheroids the lipid signal was highest in the necrotic center and lower in the inner viable cell layer.

Application of a static fluorescence-based cytometer: the CellScan in clinical immunology.

The CellScan system is a laser scanning cytometer which enables repetitive fluorescence intensity (FI) and polarization (FP) measurements in living cells, as a means of monitoring lymphocyte activation. By monitoring FP changes in peripheral blood lymphocytes (PBL) following exposure to antigenic stimuli, the CellScan may have a role in the diagnosis of autoimmune diseases. Monitoring changes in FI and FP in PBLs from patients with atherosclerosis following exposure to various stimuli, has illustrated the role of the immune system in the atherosclerotic process. The CellScan has also been evaluated as a diagnostic tool for drug-induced allergy, based on FP reduction in PBLs following incubation with the suspected drugs. FI and FP changes in cancer cells have been found to correlate with the cytotoxic effect of different anti-neoplastic drugs, illustrating the potential role of the CellScan system in clinical oncology. In conclusion, the CellScan is a promising new tool with a variety of applications in cell biology, immunology, cancer research and clinical pharmacology.

Regulation of angiogenesis by hypoxic stress: from solid tumours to the ovarian follicle.

The preovulatory follicle provides a unique physiological example of rapid growth accompanied by neovascularization, two processes that are generally characteristic of pathologies such as wound repair or malignancy. During the hours preceding ovulation, follicular growth is accompanied by elevated levels of messenger RNA for vascular endothelial growth factor (VEGF). Angiogenic activity, mediated by VEGF, is manifested in the peripheral blood vessels surrounding the follicle, that show capillary sprouting and increased vascular permeability. Following ovulation, rapid infiltration of capillaries through the follicular wall is essential for the formation of the corpus luteum. In this review we compare the preovulatory follicle with a popular model of avascular solid tumour growth, namely the multicellular tumour spheroid, in particular the role of hypoxic stress in the regulation of angiogenesis in both systems.

Cyclocreatine transport and cytotoxicity in rat glioma and human ovarian carcinoma cells: 31P-NMR spectroscopy.

Cyclocreatine (CY), an analogue of creatine, inhibits tumor growth in vivo and proliferation of tumor cells in vitro. The goal of this study was to probe the mechanism of CY transport and cytotoxicity in C6 rat glioma cells and OC238 human ovarian carcinoma cells (creatine kinase activities of 0.16 and 0.016 units/mg protein, respectively). In both cell lines, CY significantly inhibited cell growth with no effect on membrane integrity and on the content of nucleoside triphosphates. An intrinsic 31P-nuclear magnetic resonance (31P-NMR) signal of phosphocreatine, as well as accumulation of phosphocyclocreatine (PCY) after addition of CY, was observed for C6 glioma but not for the OC238 cells. Transport of CY in C6 glioma showed Michaelis-Menten kinetics for an active sodium-dependent component. Transport was reduced more than fivefold in low-glucose medium. The toxicity of CY to C6 glioma cells may be due to PCY accumulation and cellular swelling. Another mechanism must be invoked to explain CY effects on the human ovarian cancer cells in which no PCY accumulation could be detected and no cellular swelling was observed.

Modulation of water diffusion during gonadotropin-induced ovulation: NMR microscopy of the ovarian follicle.

The preovulatory rat follicle reaches a diameter of 1 mm with no internal blood vessels. Nutrient supply to the enclosed oocyte depends solely on passive diffusion across the follicular wall and the follicular fluid. Spin-echo and stimulated-echo NMR microscopy experiments were applied here for studying modulations in water diffusion during gonadotropin-induced maturation of perfused rat ovarian follicles (32 degrees C). Two diffusion compartments were observed for the follicular wall. The intracellular water diffusion coefficient, measured at a short diffusion time (9 ms) was 0.28 x 10(-5) cm2/s. Diffusion at long diffusion times was restricted to 16 microns, the size of cells in the follicular wall, and did not change during maturation. In the follicular fluid a transient 26% decrease in the diffusion coefficient was observed 4-7 h after gonadotropin stimulation, a change that is bound to affect the metabolic balance of the oocyte before ovulation.

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma.

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.