Exogenous incorporation of neugc-rich mucin augments n-glycolyl sialic acid content and promotes malignant phenotype in mouse tumor cell lines.
BACKGROUND
Carbohydrates embedded in the plasma membrane are one of the main actors involved in the communication of cells with the microenvironment. Neuraminic sialic acids are glycocalyx sugars that play important roles in the modulation of malignant cell behaviour. N-glycolylneuraminic acid (NeuGc) is synthesized by the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), an enzyme expressed in all mammals except humans. In mice, this sugar is synthesized in several somatic tissues.


METHODS
We used the B16 melanoma and F3II mammary carcinoma mouse tumor cell lines. By CMAH directed RT-PCR and NeuGc detection with the specific anti-NeuGc-GM3 antibody 14F7 we evaluated enzyme and ganglioside expression in tumor cells, respectively. Expression of NeuGc-GM3 ganglioside was reached by in vitro incubation with NeuGc-rich bovine submaxillary mucin and evaluated by slot-blot and immunohistochemistry assays using the 14F7 antibody. Tumor cells treated with mucin or purified NeuGc were injected s.c. and i.v. in syngeneic mice to evaluate tumor and metastatic growth.


RESULTS
In the present work we demonstrated the absence of expression of CMAH enzyme in B16 melanoma and F3II mammary carcinoma cells. In vitro incubation of these NeuGc-negative cells with NeuGc-rich mucin increased the presence of NeuGc in cell membranes for at least 48-72 h, as a component of the GM3 ganglioside. Preincubation with NeuGc-rich mucin reduced tumor latency and increased the metastatic potential of tumor cells in syngeneic animals. Similar results were obtained when cells were incubated with purified NeuGc alone.


CONCLUSIONS
Our results indicate that B16 and F3II mouse tumor cell lines do not express NeuGc in cell membranes but they are able to incorporate NeuGc from an exogenous source, contributing to the malignant phenotype of melanoma and mammary carcinoma cells.
Gabri
Mariano R
MR
Laboratory of Molecular Oncology, Quilmes National University, (Roque Saenz Peña 352), Bernal, (B1876BXD), Argentina. mrgabri@unq.edu.ar
Otero
Laura L
LL
Gomez
Daniel E
DE
Alonso
Daniel F
DF
D000818:Animals; D045744:Cell Line, Tumor; D005679:G(M3) Ganglioside / Q000378:metabolism; D007150:Immunohistochemistry; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009077:Mucins / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009438:Neuraminic Acids / Q000378:metabolism; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005679:G(M3) Ganglioside; D009077:Mucins; D009438:Neuraminic Acids; C032592:N-glycolylneuraminic acid; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D019158:N-Acetylneuraminic Acid