Does pH 6 beta-galactosidase activity indicate cell senescence?

Apparently two forms of beta-galactosidase (beta-GAL) in cells or tissue se ctions can be detected by enzyme histochemical staining (X-GAL). Using a se nsitive and specific HPLC method we have determined the pH dependent activi ty of beta-GAL in cell lines of lung carcinoma (A549), colon carcinoma (Cac o2-TC7), promyelocytic leukemia (HL60), hepatoma (HepG2) and human liver ho mogenates. The HPLC method has been validated and the influence of pH and s ubstrate concentration was studied. There was a good linear correlation bet ween 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 lo wer (25-50%) than pH 4.5 activity. In Caco2-TC7 cells both activities incre ased by a factor of 10 from day 3 to day 17 after seeding. In addition, sin ce 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 hardl y be considered as an exclusive 'senescence marker'. In addition, the more sensitive HPLC method could demonstrate activity in cells that showed negat ive reaction with X-GAL. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.