Does pH 6 β-galactosidase activity indicate cell senescence?

• Published in: Mechanisms of ageing and development Vol. 109; no. 2; pp. 113 - 123
• Main Authors: Krishna, Devarakonda R, Sperker, Bernhard, Fritz, Peter, Klotz, Ulrich
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 30.08.1999; Elsevier Science
• Subjects: Adult; Aged; Analytical, structural and metabolic biochemistry; beta-Galactosidase - metabolism; Biological and medical sciences; Caco-2 Cells; Cellular Senescence; Chromatography, High Pressure Liquid - methods; Enzyme histochemistry; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; HL-60 Cells; HPLC; Human cell lines; Humans; Hydrogen-Ion Concentration; Hydrolases; Liver; Middle Aged; Senescence; Tumor Cells, Cultured; β-Galactosidase; Senescence; Enzyme histochemistry; Human cell lines; Liver; β-Galactosidase; HPLC; Human; Enzyme; Lung; HPLC chromatography; Cell line; Enzymatic activity; Glycosidases; pH; Hydrolases; O-Glycosidases; Quantitative analysis
• ISSN: 0047-6374; 1872-6216
• DOI: 10.1016/S0047-6374(99)00031-7

Apparently two forms of β-galactosidase (β-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 β-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 β-GAL and pH 6 β-GAL could be demonstrated in all biological material tested and pH 6 β-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 β-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.