Abstract
Serine proteases, such as alpha-chymotrypsin or elastase, caused an aggregation of rat ascites tumour cell lines, AH-130, AH-109A and YS, in a protein free medium which preserved the cell viability. This aggregation, which was monitored spectrophotometrically, was dependent upon the protease activities and was resistant to treatment with either a calcium chelating reagent (EDTA) or neuraminidase. However, the tumour cell aggregates were redispersed by treatment with deoxyribonuclease I (DNase I). This dispersal effect was dependent upon the DNase activity. A possible relationship between the tumour cell aggregation and development of blood-borne metastasis was studied. An intravenous inoculation in rats of tumour cell aggregates performed by the alpha-chymotrypsin treatment resulted in significantly higher numbers of lung metastatic foci than an injection of single cells. When the re-separated single cells, prepared in vitro by treatment with DNase I following alpha-chymotrypsin treatment, were injected instead of the aggregates, the enhancement of metastasis was reversed. These enhancement and reversal effects were mimicked in vivo by intravenous injections of protease and nuclease following inoculation of a single cell suspension. That is, the number of metastatic foci caused by single cell inoculation followed by an intravenous alpha-chymotrypsin injection, was higher than that in a control group receiving PBS instead of alpha-chymotrypsin. Again, this augmentation was reversed by an injection of DNase I following alpha-chymotrypsin injection. Furthermore, an injection of DNase I alone itself reduced the starting number of metastases resulting from injection of the single tumour cell suspension. These data suggest that the metastatic behaviour of tumour cells may be increased by protease inducible DNA dependent cell aggregation should it occur in the blood stream.
Highlights
Crystallised bovine pancreatic a-chymotrypsin, elastase, neuraminidase, bovine muscular actin, bovine pancreatic deoxyribonuclease I (DNase I) and calf thymus DNA were purchased from Sigma Chemical Co. (St Louis, MO, USA)
AH-109A tumour cell preparations consisted of about 95% single cells, and no aggregates were observed even after tumour cells were incubated with PBS (Figure la)
Figure lb shows a typical light microscopic picture of the AH-109A tumour cell aggregates induced by a-chymotrypsin treatment
Summary
Crystallised bovine pancreatic a-chymotrypsin, elastase, neuraminidase, bovine muscular actin, bovine pancreatic DNase I and calf thymus DNA were purchased from Sigma Chemical Co. (St Louis, MO, USA). Crystallised bovine pancreatic a-chymotrypsin, elastase, neuraminidase, bovine muscular actin, bovine pancreatic DNase I and calf thymus DNA were purchased from Sigma Chemical Co. Chymostatin and elastatinal were purchased from Peptide Institute Inc. Hanks' balanced salt solution (HBSS) (without phenol red) was purchased from Nissui Pharm. (Tokyo, Japan), HBSS (without Ca2 , Mg2 + and phenol red) from Gibco (Grand Island, NY, USA). An assay kit for lactate dehydrogenase (LDH) was obtained from Shinotest Laboratories (Kanagawa, Japan)
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