Does PH 6 Beta-galactosidase Activity Indicate Cell Senescence?

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Abstract

Apparently two forms of beta-galactosidase (beta-GAL) in cells or tissue sections can be detected by enzyme histochemical staining (X-GAL). Using a sensitive and specific HPLC method we have determined the pH dependent activity of beta-GAL in cell lines of lung carcinoma (A549), colon carcinoma (Caco2-TC7), promyelocytic leukemia (HL60), hepatoma (HepG2) and human liver homogenates. The HPLC method has been validated and the influence of pH and substrate concentration was studied. There was a good linear correlation between HPLC and quantitative enzyme histochemistry (pH 4.5: r = 0.985; pH 6.0: r = 0.967). Both, pH 4.5 beta-GAL and pH 6 beta-GAL could be demonstrated in all biological material tested and pH 6 beta-GAL activity was always lower (25-50%) than pH 4.5 activity. In Caco2-TC7 cells both activities increased by a factor of 10 from day 3 to day 17 after seeding. In addition, since the beta-GAL activity decreased with increase in pH both in human liver homogenates (independent of the age of the donor) as well as in tumor cell lysates in a similar fashion we believe that the activity at pH 6 can hardly be considered as an exclusive 'senescence marker'. In addition, the more sensitive HPLC method could demonstrate activity in cells that showed negative reaction with X-GAL.

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