Ioi lle liisl lime, lle ellecls ol lysiological con-

cenlialions ol I-cainosine (-alanyl-I-lislidine) on noi-

mal luman diloid cells giown in cell culluie lave Leen
documenled. We lave used lle syslem liisl develoed Ly
HayllicI and Mooilead [1, 2], wlicl demonsliales lle
long-leim giowll ol luman liLioLlasls lollowed Ly lleii
evenlual senescence. We lave slown llal conlinuous
giowll ol llese cells in cainosine nol only incieases lleii
lilesan in oulalion douLlings and eiiod ol giowll,
Lul also can ieveise lle noimal lealuies ol senescenl
cells [3, 4]. !n ollei exeiimenls, we lave slown llal
cainosine is cyloloxic lo neolaslic cells in culluie undei
condilions in wlicl noimal cells suivive and iolileiale
[5, 6].
THL SLLSCLCL
OI H\MA D!PIO!D CLIIS
HayllicI [2] cleaily demonslialed llal luman
liLioLlasls giow al a conslanl iale, will a similai yield
ol cells ei llasI, ovei many oulalion douLlings
(PDs). Tyically, llis is in lle iange 5O-7O PDs, Lul il is
well eslaLlisled llal eaily assage amoules ol lle same
sliain lave vaiiaLle giowll olenlial [7]. Al lle end ol
exonenlial giowll, lle yield ol cells ei llasI declines,
as does lle lime iequiied loi lle oulalion lo douLle ils
size. Tle eiiod ol senescence las llen Leen ieacled.
HayllicI [2] ioosed llal llis is a manileslalion ol cel-
lulai ageing in vitro, and llal llese cells iovide excel-
lenl maleiial loi exeiimenlal invesligalions ol luman
ageing al lle cellulai level. Tlis las Leen acceled Ly
innumeiaLle laLoialoiies, wlicl ovei a eiiod ol moie
llan 3 decades lave uLlisled a veiy laige numLei ol
exeiimenlal sludies on cellulai senescence ol liLioL-
lasls, and a lessei numLei using ollei lyes ol cells,
wlicl also undeigo senescence in vitro (ieviewed in [8]).
!n view ol llese exlensive sludies will liLioLlasls, il
is suiiising llal lleie is no consensus aLoul lle genei-
al lealuies ol lleii in vitro senescence. Two sclools ol
llougll lave emeiged, and lo undeisland lle ellecls ol
cainosine on lle senescence ol luman diloid cells, il is
necessaiy lo desciiLe lle lwo dilleienl ways senescence
las Leen desciiLed in lle lileialuie. !n lleii oiiginal
sludies, HayllicI and Mooilead [1, 2] demonsliale llal
il senescenl cells aie slil wlen lley Lecome conlluenl,
lle yield ei llasI declines lo a oinl wleie lle cells no
longei ieacl conlluence. Tlese cells aie aLnoimal. Tley
aie vaiiaLle in slae and size; lley conlain excessive
gianulai maleiial and vacuoles; some cells lail lo allacl,
oi Lecome delacled, lo loim deLiis in lle medium; oll-
eis iemain allacled, Lul will nevei divide again. Al an
eailiei slage ol senescence, wlen lle cells aie aLle lo
ieacl conlluence, lley lave losl lleii aLilily lo line u in
lle aiallel aiiays wlicl is veiy claiacleiislic ol young
liLioLlasl oulalions, and lley also lave a giainy oi
gianulai aeaiance. !n sludies ovei 3O yeais, mainly
will lle diloid sliain MRC-5, all llese lealuies ol cell
senescence lave invaiiaLly Leen seen. Howevei, lleie is
anollei sclool ol llougll llal emlasizes lle lacl llal
lle iimaiy delecl in senescenl luman cells is a LlocI in
cell division. !l las liequenlly Leen slaled llal senescenl
luman cells, wlen conlluenl, can suivive loi a veiy long
eiiod iovided lle medium is clanged weeIly. Tlese
REVIEW
OOO6-2979/OO/65O7-O843$25.OO 2OOO MA!K auIa/ !nleieiiodica
* To wlom coiiesondence slould Le addiessed.
Biochemistry (Moscow), Vol. 65, No. 7, 2000, pp. 843-848. Translated from Biokhimiya, Vol. 65, No. 7, 2000, pp. 991-997.
Original Russian Text Copyright 2000 by Holliday, McFarland.
A Role for Carnosine in Cellular Maintenance
R. Holliday* and G. A. McFarland
CSIRO Division of Molecular Science, North Ryde, Sydney, N. S. . 1670, Australia;
fax. 61-2-9490-5010; E-nail. gail.ncfarlandnolsci.csiro.au or Rand1.Hollidaybigpond.con
Received ovemLei 22, 1999
AbstractTle dielide I-cainosine las Lenelicial ellecls on culluied luman liLioLlasls. Plysiological concenlialions in
slandaid media iolong lleii in vitro lilesan and sliongly ieduce lle noimal lealuies ol senescence. Iale assage cells in
noimal medium aie iejuvenaled wlen liansleiied lo medium conlaining cainosine, and Lecome senescenl wlen cainosine
is iemoved. !n lle aLsence ol yiuvale, cainosine is cyloloxic lo neolaslic and liansloimed luman and iodenl cells. one
ol llese ellecls aie seen will ils olical isomei, D-cainosine.
Key words: cainosine, ageing, luman liLioLlasls, liansloimalion
844 HOII!DAY, McIARIAD
B!OCHLM!STRY (Moscow) Vol. 65 o. 7 2OOO
cells do nol divide (oi only veiy occasionally [9]), Lul aie
aclive in RA and iolein synllesis (ieviewed in [1O]).
!l is evidenl llal lle lwo leiminal slales seen Ly dillei-
enl invesligalois aie laigely due lo lle way lle cells aie
landled. !l lley aie conlinually slil, unlil lley no
longei Lecome conlluenl, llen lle aLnoimal cells
desciiLed aLove aie seen. !l lley aie leld conlluenl and
nol slil, lley will suivive loi a long eiiod ol lime.
GROWTH OI H\MA D!PIO!D CLIIS
! MLD!\M COTA!!G CAROS!L
Human muscle conlains 2O mM cainosine, and llis
was lle concenlialion we inilially used in exeiimenls
will lelal lung sliain MRC-5, and luman loiesIin sliain
HII-1 (Iindly iovided Ly Di. R. Reddel, Clildien`s
Medical Reseaicl !nslilule, Weslmead Hosilal,
Sydney). Boll giew al lle noimal iale in MLM (Minimal
Lssenlial Medium, conlaining O.1 glucose, and no yiu-
vale) oi DMLM (DulLecco`s modilicalion ol Minimal
Lssenlial Medium, conlaining O.45 glucose and 1 mM
sodium yiuvale). Howevei, al liglei concenlialions ol
cainosine (3O mM) we lound llal lle cells giew Lellei in
DMLM, and llal llis dilleience is laigely, oi enliiely,
due lo lle addilional glucose and yiuvale. We also
lound llal only HII-1 would giow in DMLM conlain-
ing as mucl as 5O mM cainosine (aioximalely 1.25
w/v). Tlis slow giowll conlinued loi a veiy long eiiod,
as is slown in TaLle 1. Tle linal aeaiance ol llese cells
is iemaiIaLly noimal, as slown in lle liguie. Tleie aie
no gianules oi vacuoles, no deLiis in lle medium, and lil-
lle vaiiaLilily in size oi slae, wlicl is in slai conliasl
lo cells giown lo lleii linal assage in unsulemenled
DMLM. !n anollei exeiimenl, HII-1 cells weie giown
in MLM will and willoul 2O mM cainosine [3]. !n llis
case lle conliol and liealed cells giew al lle same iale loi
44 PDs, Lul lle cells giown in cainosine conlinued giow-
ing loi a lolal ol 4OO days (lasl slil al 56.8 PDs). !n con-
liasl, lle unliealed cells ceased giowll al 31O days (lasl
slil ol lwo culluies al 47.4 and 49.4 PDs).
We also liansleiied MRC-5 cells giowing in noimal
DMLM medium al PD 55.1 lo DMLM conlaining
3O mM cainosine. RemaiIaLly, llese cells conlinued
giowing slowly loi a lolal ol 413 days (lle lime ol lasl
slil), wleieas lwo conliol oulalions ceased giowll al
126 and 139 days (Iig. 5 and TaLle 1 in [3]). Ioi ieasons
unInown, ollei exeiimenls in wlicl lale assage cells
weie liansleiied liom noimal medium lo cainosine-con-
laining medium did nol give lle same sliiIing ellecl.
Tlis suggesls llal lleie may Le a ciilical eiiod duiing
lale assage giowll, Leloie wlicl cainosine las a sliong
ellecl, and allei wlicl il does nol. Iuillei exeiimenls
aie necessaiy lo invesligale llis.
!CRLASLD I!ILSPA OI CLIIS GROW
! MLD!\M W!TH CAROS!L
We lave consislenlly oLseived llal lle addilion ol
2O and 3O mM cainosine lo noimal medium incieases
Sliain
HII-1
HII-1
HII-1
MRC-5
MRC-5
MRC-5
Tiealmenl
none
3O mM
cainosine
5O mM
cainosine
none
2O mM
cainosine
3O mM
cainosine
Days ol giowll Leloie linal
slil (in aienlleses,
PDs aclieved)
391 (52.O), 432 (57.2)
522 (6O.2), 564 (63.5), 649 (61.5)
716 (48.7)
12O-15O*
315 (71.2)
321 (67)
Table 1. Lllecls ol cainosine in incieasing lle eiiod ol
giowll iioi lo leiminalion ol cell division in sliains
HII-1 and MRC-5 (all exeiimenls weie will DMLM
medium)
Tle claiacleiislic iange ol lilesans in days loi MRC-5 cells slil
inilially al 1:8, and llen al 1:4 and 1:2 in lale assages. Tle single
conliol in lle exeiimenl slown ieacled 59.8 PDs.
= >
a) HII-1 liLioLlasls giown lo lleii lasl assage in DMLM
conlaining 5O mM cainosine (llese cells lailed lo Lecome con-
lluenl); L) lle same cells allei lianslei, al aioximalely lle
same densily, lo DMLM willoul cainosine (llese cells did
nol Lecome conlluenl).
*
CAROS!L AD CLII\IAR MA!TLACL 845
B!OCHLM!STRY (Moscow) Vol. 65 o. 7 2OOO
lle lilesan ol MRC-5 and HII-1 in PDs. !n llese
exeiimenls, cainosine is iesenl liom eaily assage,
unlil giowll lully ceases. MRC-5 cells in DMLM con-
laining 2O oi 3O mM cainosine weie suLculluied al lle
same inleivals as lle conliols unlil aioximalely PD
level 45. Tleieallei lle conliol oulalions slowed
down and ieacled 56.7 and 6O.6 PDs. Tle cells in
2O mM cainosine aclieved 69.7 and 7O.7 PDs and llose
in 3O mM aclieved 64.7 and 64.3 PDs. !n exeiimenls
will HII-1 cells giown in DMLM medium, 3O mM
cainosine incieased lle lilesan Ly an aveiage ol 7.1 PDs.
Will 2O mM cainosine in MLM medium lle lilesan
incieased Ly 8.4 PDs. !n llis exeiimenl, one culluie
was giown conlinually in 2O mM D-cainosine. !l was
nol signilicanlly dilleienl liom lle conliol. !l slould Le
noled llal an inciease ol 7 PDs ieiesenls an inciease in
cell mass ol aioximalely 128 limes, and an inciease ol
1O PDs is an inciease ol aioximalely 1OOO limes. !n
anollei exeiimenl will MRC-5 cells, lle conliol o-
ulalions ieacled PD levels ol 79.2 and 76.2 [3]. Tlis is
liglly unusual and suggesls llese oulalions may lave
lad iaie clones ol long-lived cells [7], wlicl in llis case
oLscuied lle usual lile-exlension ellecl ol cainosine.
Duiing lle assaging ol luman cells, lle numLei ol
cells al conlluence is counled allei liysinizalion (using
an eleclionic Coullei counlei), and a iooilion ol llese
cells (usually
1
/
2
,
1
/
4
, oi
1
/
8
coiiesonding lo 1, 2, and 3
assages) is seeded in liesl medium inlo new llasIs.
Wlen llese cells Lecome conlluenl lley aie again counl-
ed, and liom lle giowll inciemenl lle exacl inciease in
PDs is calculaled. Tlis assumes 1OO ol lle seeded cells
allacl lo lleii suLsliale. !l was ossiLle llal cainosine
was laving an ellecl on allaclmenl, and lleieLy io-
ducing a clange in lle calculaled PDs, in comaiison lo
conliols. We lleieloie caiiied oul exeiimenls in wlicl
lle iooilion ol cells llal allacled al eveiy assage
was measuied in conliol and cainosine liealed cells.
Cainosine did nol in lacl allecl allaclmenl ol lle cells,
Lul again lleii lilesan was incieased. !n lwo exeii-
menls will MRC-5 cells in MLM and DMLM lle liles-
an was incieased Ly 11.O and 11.4 PDs, ieseclively,
wlen lle medium conlained 2O mM cainosine [4]. All
oui iesulls on lilesan exlension aie summaiized in
TaLle 2.
LIILCT OI CAROS!L
O THL PIAT!G LII!C!LCY
OI MRC-5 CLIIS
Wlen MRC-5 liLioLlasls aie laled al laiily ligl
densily, as in a 1:2, 1:4 oi 1:8 slil, lley Legin lo divide
allei aLoul 18 l. Howevei, al lowei densily, lle allacled
cells move ovei lle suilace, Lul may nol divide unlil a
small cluslei ol cells is loimed. Plaling 1O
3
eaily assage
cells in a slandaid 9-cm disl lyically yields aLoul 2O
colonies, ieiesenling a laling elliciency ol only 2.
(!l a leedei layei ol non-dividing iiiadialed oi milo-
mycin C-liealed cells is also added, lle laling ellicien-
cy is veiy mucl liglei). Wlen we laled young MRC-5
cells in DMLM conlaining 2O mM cainosine, lle laling
elliciency was veiy signilicanlly elevaled, lo aLoul 1O
[4]. Wlen 1O
3
lale assage cells aie seeded in a 9-cm disl,
no colonies aie execled Lecause lle cells lave almosl
ieacled lle end ol lleii lilesan. Howevei, cells al PD
level 56, wlen laled in DMLM conlaining 2O mM
Lxeiimenl numLei
(invesligaloi)
1 (GAM)
2 (RH)
3 (GAM)
4 (GAM)
5 (RH)
!nciease in
PDs

7.1

8.4
2.4

11.6
5.9

11.4
7.2

11.O
Table 2. !ncieases in lle lilesan in oulalion douLlings (PDs) induced Ly conlinuous giowll in medium conlain-
ing I- and D-cainosine (see [3, 4])
Iinal PD level
57.2, 52.O
6O.2, 63.5, 61.5
49.4, 47.4
56.8
5O.8
56.7, 6O.6,
7O.7, 69.7
64.7, 64.3
59.8
71.2
67.O
65.3
76.3
Tiealmenl
none
3O mM I-cainosine
none
2O mM I-cainosine
2O mM D-cainosine
none
2O mM I-cainosine
3O mM I-cainosine
none
2O mM I-cainosine
3O mM I-cainosine
none
2O mM I-cainosine
Medium
DMLM
DMLM
MLM
MLM
MLM
DMLM
DMLM
DMLM
DMLM
DMLM
DMLM
MLM
MLM
Cell sliain
HII-1
HII-1
HII-1
HII-1
HII-1
MRC-5
MRC-5
MRC-5
MRC-5
MRC-5
MRC-5
MRC-5
MRC-5
846 HOII!DAY, McIARIAD
B!OCHLM!STRY (Moscow) Vol. 65 o. 7 2OOO
cainosine, ioduced a numLei ol colonies visiLle allei
slaining will Giemsa, and none in a conliol lale. Tlese
colonies ieiesenl aLoul 15 addilional PDs. Cleaily, in
llis and ollei exeiimenls lle lale assage cells aie ieju-
venaled Ly cainosine.
RLVLRS!B!I!TY OI THL SLLSCLT
PHLOTYPL BY CAROS!L
We lave consislenlly oLseived llal lale assage
cells giown in lle iesence ol cainosine lave a moie
noimal aeaiance llan lale assage conliol cells. Tle
cainosine liealed cells line u in aiallel aiiays, lo loim
lle claiacleiislic wloils ol young noimal liLioLlasls.
Tley lacI lle lyical gianulai aeaiance ol unliealed
senescenl cells, and lleie is lillle deLiis in lle medium.
We lave also examined in many exeiimenls lle ellecl
ol liansleiiing llese lale assage cells liom cainosine
medium lo unsulemenled medium and lle ieveise
swilcl, in some cases will seveial successive swilcles
ovei a long eiiod. Tle iesulls aie lully documenled Ly
lologials in ieleiences [3, 4], and can Le summaiized
as lollows. 1) Iale assage cells will noimal moilolo-
gy in medium conlaining cainosine ieveil in 7-1O days lo
a senescenl lenolye wlen cainosine is iemoved. !n
some cases, lle senescenl lenolye Lecomes moie evi-
denl allei slilling lle cells. 2) Iale assage cells in noi-
mal medium will a senescenl lenolye aie iejuvenaled
lo a noimal lenolye wlen a ligl concenlialion ol
cainosine is added. Again, llis ellecl may Le moie cleai-
ly seen allei suLculluiing lle cells in lle iesence ol
cainosine. 3) Sequenlial lianslei ol lale assage cells lo
and liom unsulemenled and cainosine-sulemenled
medium is always accomanied Ly a swilcl in cell moi-
lology liom senescenl lo noimal, and lle ieveise. 4)
Tlese ellecls aie mosl cleaily seen in lale assage HII-
1 cells, using DMLM medium will and willoul 3O oi
5O mM cainosine. Tle liguie slows suLconlluenl HII-
1 cells in 5O mM cainosine al lleii lasl assage and lle
ellecl ol swilcling sucl cells lo unsulemenled medi-
um.
CAROS!L K!IIS TRASIORMLD
OR LOPIAST!C CLIIS
!n eaily exeiimenls, we lad one luman loiesIin
culluie llal ioved lo Le conlaminaled will liansloimed
3T3 cells. Tlese quicIly oveilooI lle giowll ol lle
luman cells. Howevei, we lad added 3O oi 5O mM
cainosine lo some llasIs, and we noliced llal in llese lle
luman cells giew will no sign ol lle 3T3 conlaminalion
cells. Ialei, we lollowed u lle oLseivalion in a seiies ol
exeiimenls will luman oi iodenl liansloimed and neo-
laslic cells [5]. We lound llal cainosine was cyloloxic lo
llese cells in MLM medium, Lul nol in DMLM medium.
Tlis dilleience was liaced lo lle 1 mM yiuvale in
DMLM, alllougl lle lowei glucose was also in ail
iesonsiLle loi lle cyloloxic ellecl ol cainosine in MLM.
We also lound llal dialyzed lelal call seium, in wlicl
low moleculai weigll comounds lave Leen iemoved,
enlanced lle cyloloxic ellecl ol cainosine. Ioi seven
liansloimed oi neolaslic luman cell lines, and lwo
iodenl lines, we delined lle cainosine-conlaining media
llal Iilled lle cells. (!n mosl cases, cyloloxicily was
oLseived as lle iounding u and delaclmenl ol lle cells,
Lul we also demonslialed lle decline in suivival ol HeIa
and CHO cells, Ly laling cainosine-liealed cells in noi-
mal medium). We also lesled all llese media, and olleis
conlaining cainosine, on noimal luman cells. !n some
lle cells giew well, Lul in olleis lle medium was cylo-
slalic lo lle cells. Tley iemained allacled lo lle suL-
sliale and lad a noimal moilology. Wlen lle caino-
sine was iemoved Ly clanging lle medium, lle liLioL-
lasls iesumed giowll. !l is veiy well Inown llal HeIa
conlaminalion quicIly iesulls in lle loss ol any conlacl-
inliLiled cell oulalion, since lle HeIa cells quicIly
ioduce oveigiowll. We devised a ioceduie wleieLy
HeIa cells conlaminaling an MRC-5 culluie aie elimi-
naled. Wlen 1O
3
HeIa cells weie added lo a oulalion
ol MRC-5 cells, allei 5 days llese HeIa cells loimed
individual colonies visiLle on a LacIgiound ol conlluenl
MRC-5 cells. Al llis lime lle cainosine liealmenl was
Legun, using MLM will 1O dialyzed seium and 2O mM
cainosine. Tle cells weie slil 3 limes in llis medium,
will inleivening clanges ol medium. Allei 27 days, no
HeIa cells weie visiLle, and lle cells weie liansleiied lo
noimal MLM. Tley giew lo lleii noimal lale assage
senescence, will no ieaeaiance ol HeIa cells [5]. Tle
same exeiimenl was also successlully caiiied oul will
ollei exeiimenlal iegimes.
We weie also inleiesled in comaiing mouse
emLiyonic slem cells (LS cells) will mouse emLiyonic
leialocaicinoma cells (LC cells). Tlese aie Loll immoi-
lal luiiolenl cell lines, Lul LS cells lave a noimal
luiiolenl lenolye. As in lle case ol diloid somalic
cells and liansloimed cells, we lound llal cainosine ie-
venls lle giowll ol lle LC cells in lle aLsence ol yiu-
vale, wleieas lle LS cells giow well undei lle same con-
dilions. Tlus, lle neolaslic lenolye is again associ-
aled will cainosine sensilivily [6].
As well as yiuvale, we also clecIed lle ellecl ol
ollei comonenls ol lle liicaiLoxylic cycle in nullilying
lle cyloloxic ellecls ol cainosine. We lound llal 1 mM
oxaloacelale oi 1 mM -Ieloglulaiale lad an ellecl com-
aiaLle lo yiuvale, Lul ciliale, isociliale, succinale,
lumaiale, and malale lad no ellecl. We also demonslial-
ed llal D-cainosine in MLM willoul yiuvale is enliiely
nonloxic lo HeIa cells. We lesled lomocainosine, ansei-
ine, -alanine, and I-lislidine on HeIa cells. Only ansei-
ine las cyloloxic aclivily liIe cainosine (i.e., deendenl
CAROS!L AD CLII\IAR MA!TLACL 847
B!OCHLM!STRY (Moscow) Vol. 65 o. 7 2OOO
on lle aLsence ol yiuvale), and 2O mM lislidine is loxic
lo HeIa cells iiieseclive ol lle iesence ol yiuvale [5].
D!SC\SS!O
Ioi sucl a small molecule, il is iemaiIaLle llal
cainosine las mullile ioeilies. !l is an anlioxidanl, a
clelaloi ol loxic melal ions, a Lullei, and an inliLiloi ol
non-enzymic glycosylalion ol ioleins. Tlese ioeilies
aie ieviewed and discussed elsewleie in llis volume. !ls
ligl concenlialion in sIelelal muscle suggesls llal il
may lave a iole in neulializing lle accumulalion ol lac-
lic acid, wlicl occuis wlen muscles oLlain eneigy liom
glycolysis iallei llan iesiialion. !l is, lowevei, sliiIing
llal luman muscle conlains 2O mM cainosine, wleieas
mouse muscle conlains only 1 mM ([11], 1. Miclaelis,
eisonal communicalion). Tlis suggesls llal cainosine
may lave a moie lundamenlal iole in cellulai mainle-
nance, since il is well eslaLlisled llal lle elliciency ol
mainlenance in mammalian secies is coiielaled will
lilesan [8, 12]. Tle ioosed mainlenance lunclion
could include anlioxidanl aclivily, clelalion ol loxic
melals, and lle inliLilion ol non-enzymic glycosylalion
ol ioleins and lle accumulalion ol AGLs (advanced
glycalion end ioducls). Tlese ligl-moleculai-weigll
aggiegales aie seen in seveial lissues in senescenl ani-
mals and oLviously accumulale moie iaidly in sloil-
lived animals llan in long-lived ones.
Tle ellecl ol cainosine in ieveising lle noimal
symloms ol cell senescence, and also incieasing cells`
giowll olenlial, may well Le a ielleclion ol mainle-
nance lunclions. !l is widely Lelieved llal lle linal ces-
salion ol cell division allei lle iolonged giowll ol
diloid somalic cells is due lo lle induclion ol a LlocI in
cell division (ieviewed in [13, 14]). Tle iolein 53 las a
cenlial iole as guaidian ol lle genome. Tlis means
llal wlen DA damage occuis, cell division is LlocIed
lliougl lle 53-medialed inliLilion ol DA synllesis.
One lye ol DA damage llal las ieceived a gieal deal
ol allenlion is lle sequenlial loss ol lelomeiic DA.
Tlis loss evenlually liiggeis a iesonse involving a LlocI
in cellulai division. Howevei, in llis case, lleie is no
iecoveiy liom lle damage, so lle cell nevei divides
again. !l is also ossiLle llal ollei lyes ol clange in
DA lave lle same ellecl, loi inslance, lle accumula-
lion ol delecls in DA sliucluie, oi lle loss ol DA
mellylalion. Cainosine could oeiale in one ol lwo
ways. Iiisl, il could ieduce lle degiee oi incidence ol lle
evenls llal evenlually liiggei lle cell cycle LlocI. Ioi
examle, il could ieduce lle lengll ol lle lelomeiic
DA liagmenls losl, oi lle iale ol DA mellylalion
decline. Second, il could ieduce lle lysiological ellecls
ol clanges in DA, so llal lle oinl al wlicl a linal cell
division LlocI occuis is signilicanlly delayed. Sucl an
ellecl would manilesl ilsell in lle iejuvenalion ol lle cel-
lulai lenolye, wlicl is seen wlen senescenl cells aie
giown in oi liansleiied lo a medium conlaining caino-
sine. Similaily, lle iemoval ol cainosine would iesull in
lle ieveision ol a laiily noimal cellulai lenolye lo a
visiLly aLnoimal senescenl cell. OLviously, a numLei ol
exeiimenlal aioacles lo lesl oi dislinguisl llese os-
siLililies would now Le ossiLle.
Tle dilleienlial ellecl ol cainosine on noimal cells
and lleii liansloimed deiivalives is exliemely sliiIing.
!l is ossiLle llal cainosine las anli-cancei aclivily in
vivo. Tlis would ceilainly lil in will lle well-Inown lacl
llal luman cells aie mucl less ieadily liansloimed llan
iodenl cells [12], and lle lwenly-lold liglei concenlia-
lion ol cainosine in some luman cells, sucl as llose in
sIelelal muscle, llan in lle same mouse cells.
!l was discoveied Ly WaiLuig many decades ago
llal cancei cells lave mucl liglei glycolysis aclivily
llan noimal cells [15]. He llougll cancei cells weie
deleclive in iesiialion, wlicl is nol coiiecl, Lul il las
Leen exliemely well eslaLlisled llal lle glycolysis/iesi-
ialion ialio in lle geneialion ol ATP is lai liglei in can-
cei cells llan noimal somalic cells [16]. !n ollei woids,
liansloimed oi neolaslic cells lave uncouled lle noi-
mal iegulalion ol glycolysis and iesiialion. Will iegaid
lo cainosine, il is well Inown llal il ieacls Ly lle
Amadoii ieaclion will aldelyde and Ielo gious [17]. !l
is ailiculaily ieaclive will lle losloiylaled liiose
sugai glycolysis inleimediales. Tlis immedialely sug-
gesls llal cainosine inliLils glycolysis aclivily and
lleieloie lle geneialion ol ATP in cancei cells. Tle
ellecl is lolally ieveised Ly lle addilion ol yiuvale (and
al leasl lwo ollei KieLs cycle inleimediales). Tlese
would ol couise slimulale iesiialion lo geneiale ATP,
and allow lle lumoi cells lo giow noimally. !l is also sig-
nilicanl llal anollei clemical llal ieacls will sugais,
Inown as lenilselam, also inliLils HeIa cell giowll in
lle aLsence ol yiuvale, Lul nol in ils iesence [5]. !n
addilion, il is liglly signilicanl llal we lave slown llal
cainosine las dilleienlial ellecls on LS (emLiyonic
slem) cells and LC (leialocaicinoma) cells. Boll llese
lyes ol cells aie immoilal cell lines, and Loll aie
luiiolenl in lleii caacily lo dilleienliale inlo many
lyes ol cell and lissue. Yel only lle LC cells aie inliL-
iled Ly cainosine in lle aLsence ol yiuvale [6].
Mucl iecenl ieseaicl on lle moleculai Liology ol
cell liansloimalion and immoilalizalion comlelely
ignoies lle lundamenlal dilleiences in lle lysiology
and Lioclemisliy ol cancei cells and noimal cells. Aail
liom glycolysis and iesiialion, il las long Leen Inown
llal lleie can also Le iolound dilleiences in lle aclivi-
lies ol many imoilanl melaLolic enzymes in cancei and
noimal cells (ieviewed in [18]; loi a iecenl examle, see
[19]). Tleie is mucl documenlalion ol mulalions in
oncogenes and lumoi suiessoi genes llal iedisose
oi cause noimal cells lo Lecome malignanl. !l slould nol
Le loigollen llal lle swilcl liom a noimal LS cell lo an
848 HOII!DAY, McIARIAD
B!OCHLM!STRY (Moscow) Vol. 65 o. 7 2OOO
aLnoimal LC cell does nol deend on gene mulalions.
!nseilion ol a mouse emLiyo inlo an aLnoimal enviion-
menl (sucl as llal Leneall an adull Iidney casule) is
sullicienl lo geneiale leialocaicinomas. Tlis is a leiila-
Lle eigenelic evenl, wlicl cleaily las lundamenlal
ellecls on lle iegulalion ol cellulai melaLolism.
Cainosine may Le a veiy uselul lool lo ioLe luillei nol
only lle lysiological dilleiences Lelween noimal and
lumoi cells, Lul also lle imoilance ol cellulai mainle-
nance in delaying ageing oi iolecling cells againsl
oncogenic liansloimalion.
RLILRLCLS
1. HayllicI, I., and Mooilead, P. S. (1961) Exp. Cell Res.,
25, 585-621.
2. HayllicI, I. (1965) Exp. Cell Res., 37, 614-636.
3. McIailand, G. A., and Holliday, R. (1994) Exp. Cell Res.,
212, 167-175.
4. McIailand, G. A., and Holliday, R. (1999) Exp. Geront.,
34, 35-45.
5. Holliday, R., and McIailand, G. A. (1996) Brit. J.
Cancer, 73, 966-971.
6. McIailand, G. A., and Holliday, R. (1999) In vitro Cell
Devel. Biol. Aninal, 35, 15-16.
7. Holliday, R., Huscllscla, I. !., Taiianl, G. M., and
KiiIwood, T. B. I. (1977) Science, 198, 366-372.
8. Holliday, R. (1995) Understanding Ageing, CamLiidge
\niveisily Piess, CamLiidge.
9. Malsumuia, T., Zeiiudo, Z., and HayllicI, I. (1979) J.
Gerontol., 34, 328-334.
1O. Smill, 1. R., and Iincoln, D. W. (1984) Int. Res. Cytol.,
89, 151-177.
11. Mannion, A. I., 1aIeman, P. M., Dunnell, M., Haiiis, R.
C., and Willian, P. I. (1992) Eur. J. Appl. Physiol., 64, 47-
5O.
12. Holliday, R. (1996) in Genetic Instability in Cancer
(Iindall, T., ed.) Cold Siing HaiLoi IaLoialoiy Piess,
. Y., . 1O3-115.
13. Reddel, R. (1998) BioEssays, 20, 977-984.
14. Wynloid-Tlomas, D. (1999) J. Pathol., 187, 1OO-111.
15. WaiLuig, O. (1956) Science, 123, 3O9-314.
16. AisenLeig, A. C. (1961) The Glycolysis and Respiration of
Tunors, Academic Piess, . Y.
17. HiIiss, A. R. (2OOO) Biochenistry ,Moscow), 65, 771-
778.
18. WeLei, G. (1983) Cancer Res., 43, 3466-3492.
19. Holliday, R., and Ho, T. (1998) Proc. Natl. Acad. Sci.
USA, 95, 8727-8732.