Louis, MO, USA). NSC 683864 solubility dmso After a 3-h incubation, the supernatant was discarded, cells were resuspended in DMSO and absorbance was measured at 570 nm. In vivo inoculation of BSM or NeuGc-preincubated cells into syngeneic mice Tumor cell suspensions were preincubated with 500 μg/ml of BSM or 100 μg/ml of NeuGc in culture medium for 1 h and then extensively washed and resuspended. Control cells were incubated in the same medium without the addition of BSM or NeuGc. Inbred C57BL/6 and Balb/c mice were inoculated intravenously
with 1 × 105 B16 and F3II cells, respectively. After 22 days, lungs were collected, fixed in Bouin’s solution, and metastasic foci were counted under a dissecting microscope. In another set of experiments, mice were injected subcutaneously with B16 tumor cells preincubated or not with BSM. The time of appearance of local tumors was monitored by palpation and further confirmed by histopathology. Tumor size was measured
with a caliper twice a week and tumor diameter was calculated as the square root of width × length. Animals were sacrificed 60 days after tumor inoculation or when they became moribund. Results We first checked the expression of CMAH in B16 melanoma and F3II mammary carcinoma cells. To assess the presence of CMAH mRNA, an RT-PCR assay using high affinity primers was performed. As expected, normal liver was positive for CMAH expression, but neither B16 nor F3II cells expressed the gene. When performed on total RNA from normal liver, the RT-PCR assay yielded 3 distinct products (Fig. 1). After sequencing, all 3 GSK458 datasheet shared a very high homology with the CMAH gene sequence. The intermediately-sized amplicon shared a 99% LY294002 molecular weight identity with the CMAH sequence while the other two proved to be alternatively spliced variants, as reported by Koyama et al . Figure 1 Expression of the CMAH mRNA evidenced by RT-PCR. Lane 1, total RNA from
the B16 mouse melanoma cell line; lane 2, total RNA from the F3II mouse mammary carcinoma cell line; lane 3, total RNA from normal mouse liver. We then examined the expression of NeuGc in tumor cells by immunohistochemical staining, using the 14F7 antibody reactive against NeuGc-GM3. No expression was detected under serum-free in vitro culture conditions. On the contrary, in the presence of FBS both B16 and Thiamine-diphosphate kinase F3II cells became clearly positive (Fig. 2A-D), suggesting that NeuGc can be incorporated from the bovine source. Figure 2 Indirect immunoperoxidase staining of the NeuGc-GM3 ganglioside with 10 μg/ml of 14F7 monoclonal antibody on formalin-fixed B16 (A, B and E) and F3II (C, D and F) monolayers, cultured in the presence (B and D) or absence (A and C) of 10% FBS or incubated with 250 μg/ml mucin in FBS-free medium for 24 h (E and F). Original magnification 1000×. In order to increase NeuGc density in the cell membrane, we incubated B16 and F3II cells in vitro with the minor type of BSM, a mucin fraction with high NeuGc content .