Bo Yun Lee¹, Jung A. Han¹, Jun Sub Im¹, Amelia Morrone², Kimberly Johung³, Edward C. Goodwin³, Wim J. Kleijer⁴, Daniel DiMaio^3,5,*, Eun Seong Hwang^1,3,*

¹ Department of Life Science, University of Seoul, Dongdaemungu, Jeonnongdong, Seoul, Korea 130-743
² Metabolic and Muscular Disease Unit, Department of Pediatrics, University of Florence, Florence, Italy
³ Department of Genetics, Yale University School of Medicine, New Haven, CT 06520-8005, USA
⁴Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands
⁵ Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520-8005, USA

^*Correspondence Daniel DiMaio, Department of Genetics, Yale University School of Medicine, PO Box 208005, New Haven, CT 06520-8005, USA.
Eun Seong Hwang, Department of Life Science, University of Seoul, Dongdaemungu, Jeonnongdong, Seoul, Korea 130-743.

Summary
Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence-associated β-galactosidase (SA-β-gal), which is defined as β-galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA-β-gal and its cellular roles in senescence are not known. We demonstrate here that SA-β-gal activity is expressed from GLB1, the gene encoding lysosomal β-D-galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive G_M1-gangliosidosis, which have defective lysosomal β-galactosidase, did not express SA-β-gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small-hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA-β-gal. GLB1 mRNA depletion also prevented expression of SA-β-gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA-β-gal induction during senescence was due at least in part to increased expression of the lysosomal β-galactosidase protein. These results also indicate that SA-β-gal is not required for senescence.

Keywords: aging; cellular senescence; gene expression; GLB1; HeLa cells; lysosomes; molecular biology of aging; senescence