Does pH 6 beta-galactosidase activity indicate cell senescence?
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.
Krishna
D R
DR
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
Sperker
B
B
Fritz
P
P
Klotz
U
U
D000328:Adult; D000368:Aged; D018938:Caco-2 Cells; D016922:Cellular Senescence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018922:HL-60 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008875:Middle Aged; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism*
D001616:beta-Galactosidase