Senescence-associated (beta)-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal (beta)-galactosidase. Here we have investigated the origin of senescence-associated-(beta)-galactosidase activity by modifying the intracellular and lysosomal pH of young and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytometric assay. Lysosomal alkalinisation with chloroquine or bafilomycin A(1), as well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular (beta)-galactosidase activity. However, independent of pH alterations, senescent cells showed levels of (beta)-galactosidase activity three- to sixfold higher than young cells. This increase in activity occurred in parallel to an increase in (beta)-galactosidase protein levels. Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated with the increase in (beta)-galactosidase. These findings demonstrate that senescence-associated (beta)-galactosidase is a manifestation of residual lysosomal activity at a suboptimal pH, which becomes detectable due to the increased lysosomal content in senescent cells.

Atherton
A. J.
,
O'Hare
M. J.
,
Buluwela
L.
,
Titley
J.
,
Monaghan
P.
,
Paterson
H. F.
,
Warburton
M. J.
,
Gusterson
B. A.
(
1994
).
Ectoenzyme regulation by phenotypically distinct fibroblast sub-populations isolated from the human mammary gland.
J. Cell Sci
107
,
2931
2939
Ben-Yosseph
Y.
,
Shapira
E.
,
Edelmann
E.
,
Burton
B. K.
,
Nadler
H. L.
(
1977
).
Purification and properties of neutral-galactosidase from human liver.
Arch. Biochem. Biophys
184
,
373
380
Bodnar
A. G.
,
Ouellette
M.
,
Frolkis
M.
,
Holt
S. E.
,
Chiu
C.
,
Morin
G. B.
,
Harley
C. B.
,
Shay
J. W.
,
Lichtsteiner
S.
,
Wright
W. E.
(
1998
).
Extension of life-span by introduction of telomerase into normal human cells.
Science
279
,
349
352
Bosmann
H. B.
,
Gutheil
R. L.
,
Case
K. R.
(
1976
).
Loss of critical neutral protease in ageing WI-38 cells.
Nature
261
,
499
501
Brunk
U. T.
,
Ericsson.
J. L. E.
,
Ponten
J.
,
Westermark
B.
(
1973
).
Residual bodies and ‘aging’ in cultured human glia cells.
Exp. Cell Res
79
,
1
14
Campisi
J.
(
1996
).
Replicative senescence: an old lives' tale?.
Cell
84
,
497
500
Cristofalo
V. J.
,
Kabakjian
J.
(
1975
).
Lysosomal enzymes and aging in vitro: subcellular enzyme distribution and effect of hydrocortisone on cell life-span.
Mech. Ageing Dev
4
,
19
28
d'Azzo
A.
,
Hoogeveen
A. T.
,
Reuser
A. J. J.
,
Robinson
D.
,
Galjaard
H.
(
1982
).
Molecular defect in combined-galactosidase and neuraminidase deficiency in man.
Proc. Natl. Acad. Sci. USA
79
,
4535
4539
Devarakonda
R. K.
,
Sperker
B.
,
Fritz
P.
,
Klotz
U.
(
1999
).
Does pH 6-galactosidase activity indicate cell senescence?.
Mech. Ageing Dev
109
,
113
123
Dimri
G. P.
,
Lee
X.
,
Basile
G.
,
Acosta
M.
,
Scott
G.
,
Roskelly
C.
,
Medrano
E. E.
,
Linskens
M.
,
Rubelj
I.
,
Pereira-Smith
O.
,
Peacocke
M.
,
Campisi
J.
(
1995
).
A biomarker that identifies senescent human cells in culture and in aging skin in vivo.
Proc. Natl. Acad. Sci. USA
92
,
9363
9367
Escobales
N.
,
Longo
E.
,
Cragoe
E. J.
,
Danthuluri
N. R.
,
Brock
T. A.
(
1990
).
Osmotic activation of Na+-H+exchange in human endothelial cells.
Am. J. Physiol
259
,
640
–.
Fiering
S. N.
,
Roederer
M.
,
Nolan
G. P.
,
Micklem
D. R.
,
Parks
D. R.
,
Herzenberg
L. A.
(
1991
).
Improved FACS-Gal: Flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs.
Cytometry
12
,
291
301
Goldstein
S.
,
Harley
C. B.
(
1979
).
In vitro studies of age-associated diseases.
Fed. Proc
38
,
1862
1867
Hartz
P. A.
,
Wilson
P. D.
(
1997
).
Functional defects in lysosomal enzymes in autosomal dominant polycystic kidney disease (ADPKD): abnormalities in synthesis, molecular processing, polarity, and secretion.
Biochem. Mol. Med
60
,
8
26
Hayflick
L.
,
Moorhead
P. S.
(
1961
).
The serial cultivation of human diploid cell strains.
Exp. Cell Res
25
,
585
621
Hayflick
L.
(
1965
).
The limited in vitro life time of human diploid cell strains.
Exp. Cell Res
37
,
614
636
Hayflick
L.
(
1980
).
Cell Aging.
Ann. Rev. Gerontol. Geriatr
1
,
26
67
Ho
M. W.
,
Cheetham
P.
,
Robinson
D.
(
1973
).
Hydrolysis of GM1-ganglioside by human liver-galactosidase isoenzymes.
Biochem. J
136
,
351
359
Hoogeveen
A. T.
,
Verheijen
F. W.
,
Galjaard
H.
(
1983
).
The relation between human lysosomal-galactosidase and its protective protein.
J. Biol. Chem
258
,
12143
12146
Matsunaga
H.
,
Handa
J. T.
,
Aotaki-Keen
A.
,
Sherwood
W.
,
West
M. D.
,
Hjelmeland
L. M.
(
1999
).
-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells.
Invest. Ophthalmol. Vis. Sci
40
,
197
202
Meisler
M.
,
Rattazzi
M. C.
(
1974
).
Immunological studies of-galactosidase in normal human liver and in GM1 gangliosidosis.
Am. J. Hum. Genet
26
,
683
691
Mishima
K.
,
Handa
J. T.
,
Aotaki-Keen
A.
,
Lutty
G. A.
,
Morse
L. S.
,
Hjelmeland
L. M.
(
1999
).
Senescence-associated-galactosidase histochemistry for the primate eye.
Invest. Ophthalmol. Vis. Sci
40
,
1590
1593
Morreau
H.
,
Galjart
N. J.
,
Gillemans
N.
,
Willemsen
R.
,
van der Horst
G. T. J.
,
d'Azzo
A.
(
1989
).
Alternative splicing of-galactosidase mRNA generates the classic lysosomal enzyme and a -galactosidase-related protein.
J. Biol. Chem
264
,
20655
20663
Negulescu
P. A.
,
Machen
T. E.
(
1990
).
Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes.
Methods Enzymol
192
,
38
81
Norden
A. G.
,
Tennant
L. L.
,
O'Brien
J. S.
(
1974
).
GM1ganglioside-galactosidase. A. Purification and studies of the enzyme from human liver.
J. Biol. Chem
249
,
7969
7976
Reznikoff
C. A.
,
Yaeger
T. R.
,
Belair
C. D.
,
Saveliena
E.
,
Phthenveettil
J. A.
,
Stadler
W. M.
(
1996
).
Elevated p16 at senescence and loss of p16 at immortalization in human papillomavirus 16 E6, but not E7, transformed human uroepithelial cells.
Cancer Res
56
,
2886
2990
Robbins
E.
,
Levine
E. M.
,
Eagle
H.
(
1970
).
Morphologic changes accompanying senescence of cultured human diploid cells.
J. Exp. Med
131
,
1211
1222
Rudolph
K. L.
,
Chang
S.
,
Millard
M.
,
Schreiber-Agus
N.
,
DePinho
R. A.
(
2000
).
Inhibition of experimental liver cirrhosis in mice by telomerase gene delivery.
Science
287
,
1253
1258
Serrano
M.
,
Lin
A. W.
,
McCurrach
M. E.
,
Beach
D.
,
Lowe
S. W.
(
1997
).
Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.
Cell
88
,
593
602
Sigal
S. H.
,
Rajvinshi
P.
,
Gorla
G. R.
,
Sokhi
R. P.
,
Saxena
R.
,
Gebhard
D. R.
Jr.
,
Reid
L. M.
,
Gupta
S.
(
1999
).
Partial hepatectomy-inducedpolyploidy attenuates hepatocyte replication and activates cell aging events.
Am. J. Physiol
276
,
1260
–.
Smith
J. R.
,
Pereira-Smith
O. M.
(
1996
).
Replicative senescence: implications for in vivo aging and tumor suppression.
Science
273
,
63
67
Tsukamoto
A.
,
Kaneko
Y.
,
Yoshida
T.
,
Han
K.
,
Ichinose
M.
,
Kimura
S.
(
1998
).
2-Methoxyestradiol, and endogenous metabolite of estrogen, enhances apoptosis and-galactosidase expression in vascular endothelial cells.
Biochem. Biophys. Res. Commun
248
,
9
12
Turk
B.
,
Milo
G. E.
(
1974
).
An in vitro study of senescent events of human embryonic lung (WI-38) cells.
Arch. Biochem. Biophys
161
,
46
53
van der Loo
B.
,
Fenton
M. J.
,
Erusalimsky
J. D.
(
1998
).
Cytochemical detection of a senescence-associated-galactosidase in endothelial and smooth muscle cells from human and rabbit blood vessels.
Exp. Cell Res
241
,
309
315
Yegorov
Y.
,
Akimov
S. S.
,
Hass
R.
,
Zelenin
A. V.
,
Prudovsky
I. A.
(
1998
).
Endogenous-galactosidase acitivity in continuously nonproliferating cells.
Exp. Cell Res
243
,
207
211
Yin
D.
(
1996
).
Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores.
Free Rad. Biol. Med
21
,
871
888
Yoshimori
T.
,
Yamamoto
A.
,
Moriyama
Y.
,
Futai
M.
,
Tashiro
Y.
(
1991
).
Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degredation in lysosomes of cultured cells.
J. Biol. Chem
266
,
17707
17712
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