Japanese Journal of Oral and Maxillofacial Surgery
Online ISSN : 2186-1579
Print ISSN : 0021-5163
ISSN-L : 0021-5163
Sialic acid demonstration on human oral cancer cells in primary culture using enzyme treatment and ultrastructural binding of Limulus polyphemus agglutinin
Takashi SAKAMOTO
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1990 Volume 36 Issue 11 Pages 2458-2472

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Abstract

The localization of sialic acid on human oral cancer cells was compared with that on normal cells. Primary culture cells from 16 samples of squamous cell carcinomas and 4 samples of normal gingival epithelia were used.
HeLa and SCC-4 cell lines were also studied as a positive control. All of these cells were grown on plastic dishes in a culture medium containing 20% fetal bovine serum. Morphological changes of HeLa or SCC-4 cells were examined after treatment with neuraminidase (NAase), chondroitinase ABC (CHase) and hyaluronidase (HAase). Furthermore the combined effects of these enzymes on the cell morphology were determined. Oral cancer cells were treated with CHase-NAase.
For HRP-LPA stain, the culture cells were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde, and were immersed in HRP-LPA (horseradish peroxidase-Limulus polyphemus agglutinin) conjugate for 60 min. The cells were developed with 3, 3'-diaminobenzidine and processed by a routine method for electron microscopy. As a control study to confirm thebinding specificity of LPA, the cells were treated with HRP-LPA conjugate combined with 0.2M inhibitory sugar. The cells were also treated with HRP-LPA after pretreatment with 0.02N sulfuric acid or NAase.
Both HeLa and SCC-4 cells were detached from dishes after treatment with NAase-CHase. A most intensive change of cell form was observed when the cells were treated with CHase-NAase. These results suggested that sialic acid on the cell surface was removed effectively by CHase-NAase treatment. Eight out of 16 cases of oral cancer showed changes of cell form by this treatment. The cells in 3 cases all became round and were detached from the plastic surface. On the contrary, normal cells horn the gingival epithelium did not change by CHase-NAase treatments.
Surfaces of HeLa and SCC-4 cells were strongly stained with HRP-LPA. The LPA labelling intensity was diminished by the pretreatment with inhibitory sugar or sulfuric acid.
NAase digestion also reduced the labelling intensity. Although LPA staining was seen on the surface of all kinds of cancer cells, the intensity differed with the case. None of the normal cells were stained with HRP-LPA. The results of HRP-LPA staining and enzyme treatment revealed that the cases showing intensive changes by enzyme treatment also showed a strong staining with HRP-LPA.
In conclusion, oral cancer cells in the primary culture possessed more sialic acid on the cell suface than normal cells, and the content of sialic acid on cancer cells differed with the case.

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© Japanese Society of Oral and Maxillofacial Surgeons
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