Production of a highly cytopathic HIV-1 isolate from a human mucosal epithelial cell line cultured on microcarrier beads in serum-free medium.

The human colonic epithelial cell line HT-29 can be productively infected with various HIV-1 and HIV-2 isolates that are highly cytopathic for T lymphocytes. In each case, a chronically infected HT-29 cell line can be established, and progeny viruses retain their original properties including high cytopathogenicity for T cells. Inasmuch as AIDS vaccines should include viral isolates capable of infecting mucosal epithelial cells, it may be useful to produce these isolates in such cells at a large scale. We describe here a microcarrier-based culture system allowing the production of infectious viruses from HT-29 cells grown in a chemically defined serum-free medium (Dulbecco's modified Eagle's medium/F12, HEPES 15 mM, pH 7.4, transferrin 5 micrograms/ml, selenium 10 ng/ml). The yield of HIV-1 from microcarrier cultures (275 ng of p24gag/ml) was greater than the yield from conventional culture flasks (122 ng of p24gag/ml). This virus, produced in serum-free medium, can be used either as a viral stock or as a source for HIV-1 proteins.

Energetic metabolism of glucose, mannose and galactose in glucose-starved rat insulinoma cells anchored on microcarrier beads. A phosphorus-31 NMR study.

Insulin-secreting cells (RINm5F) have successfully been grown on a large scale on poly-L-lysine coated-polystyrene microcarriers, providing a high cell number in a restricted volume under conditions that respect the metabolic integrity of these anchorage-dependent cells. The energetic metabolism of the perfused cells has been followed non-invasively by phosphorus-31 nuclear magnetic resonance spectroscopy. Glucose starvation induced a rapid decrease in nucleoside triphosphates (mainly ATP) pools, correlated with an increase in Pi level. The initial ATP level was rapidly recovered when the cells were refed with glucose or with mannose, but not with galactose, even after 2 h of perfusion. These differential effects of hexoses on energetic metabolism might be related to their various insulin-release actions on tumor islet cells.

Suramin inhibits proliferation of rat glioma cells and alters N-CAM cell surface expression.

Suramin, a drug used in the treatment of trypanosomiasis and onchocerciasis inhibits growth-factor-induced mitogenesis. We have investigated the effect of suramin on the growth rate and the morphology of C6 glioma cells cultured in the presence of serum or in a serum-free defined medium. Exponentially growing cells were seeded in multi-dish plates (5 x 10(4) cells/2 cm2 well) in DMEM supplemented with 5% fetal calf serum and were continuously exposed to 1 microgram/ml to 1,000 micrograms/ml suramin. Growth rate (determined 9 days after seeding) was reduced by 5%, 33%, 56% and 97%, respectively for suramin concentrations of 1, 10, 100 and 1000 micrograms/ml. Similar results were obtained in serum-free defined medium (DMEM/F12, 1:1, v:v, EGF 5 ng/ml, transferrin 5 micrograms/ml, selenium 10 ng/ml). Moreover, the concentration of suramin in the culture medium remained constant, demonstrating that the drug was not actively metabolized by the cells. Suramin also induced marked changes in cell morphology: the usual bipolar shape of C6 cells evolved toward a more differentiated appearance, with numerous cellular processes allowing a wide number of cell-cell contacts. In parallel, we monitored expression of an adhesion molecule (N-CAM) at both the mRNA and protein levels. Indirect immunofluoresence technique showed an important increase in cell surface N-CAM expression, starting from a dose of 10 micrograms/ml suramin, whereas total cellular content of N-CAM protein as well as its mRNA levels were unaffected. We also observed that the levels of expression of actin and N-CAM mRNAs decreased by a factor of two in cells maintained in defined medium. However, the relative ratio of N-CAM mRNA over actin mRNA was virtually unchanged following suramin treatment. Taken together, our results suggest that suramin (i) exerts a blocking effect of autocrine growth factors, (ii) interferes with the turn-over mechanisms of N-CAM expressed at the cell surface, either by impairing its endocytosis and/or the process of release of the N-CAM 120 isoform.