Standard and quantitative analysis of cyclin E threshold by cyclin E/DNA multiparameter flow cytometry.

Abstract

The threshold of cyclin E expression at G1/S boundary is a characteristic feature of cell cycle progressing. In this study, we tried to develop a quantitative approach to analyze cyclin E threshold by multiparameter flow cytometry. The expression of cyclin E in exponentially growing MOLT-4 cells was detected under different photomultiplier tube (PMT) voltages by cyclin E/DNA multiparameter flow cytometry. Additionally, cyclin E was detected in cells which were treated with caffeine and cycloheximide (CHX) under the same PMT voltage. Moreover, the expression of cyclin E in MOLT-4 cells was compared with that in JURKAT cells. Cyclin E threshold was quantified by formula B2/AxC (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity, respectively). Results showed that in MOLT-4 cells, cyclin E threshold calculated by formula B2/AxC was invariable under different PMT settings. It was decreased in cells treated with caffeine and remained changeless in cells treated with cycloheximide. Cyclin E threshold in JURKAT cells was much lower than that in MOLT-4 cells. It was suggested that Formula B2/AxC we firstly set up could be used to analyze cyclin E expression threshold quantitatively.

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282
of Scienc e and
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Standard and Quantitative A nalysis of Cyclin E T hreshold by Cyclin
E ／ DNA M ultiparameter Flow Cytometry
X IE Daxing ( 谢 大兴 ) ， F E NG Y ongdong ( 冯永 东) ， W U J ianhong ( 吴剑宏 ) ， L IU Shuangyou ( 刘 双又) ，
L I X iaolan ( 李小兰) ，T A O Deding ( 陶德定) ，GONG J ianping ( 龚建 平)
T P Ca cPr R PsPnrfh I nstitute，T ongj i H ospital ，T ongj i M edical Col leg e，H uazhong University of S( ience and T echnology ，
W “ ha n 4 30 03 0 。C hi na
Sum m ary ：T he thresho ld o f cy cli n
cy cle p ro g ressin g． In th i s stud y ，
thr esho ld by m uhi pa ram ete r f low cy to m etry ． T he ex pr essio n of cyc lin E i n ex po ne nti all y g ro wi ng
M0 I T 一 4 cells w as detec ted under different photomuhiplier tube ( P M T ) voltages by cyclin E ／ DN A
ITIUhi par am eter flo w cy to m etr y． A ddi tio nal ly 。 cy cli n E w as dete cte d i n ce ll s whi ch wer e trea ted
wi th caffeine and cy clohe xim ide ( C H X ) unde r the sam e P M T v ol tag e．M o reo ver ，the ex pr essi o n of
cvclin E in M () L T 一 4 cells w as compared w ith that in J U R K A T cells．C yclin E thresho ld w as quan—
t ified by formula B ／ A × C ( A 。B。C indicates t he minimum，threshold ，and maximum of cyclin E
flno rescence intensi ty ，r espectiv el y ) ．R esult s show ed tha t i n M O I T 一 4 ce ll s，cycli n E thresho ld ca l-
c ulated by formula B ／ A × C was invariable under different P M T set tings．It was decreased in cells
trea ted w i th ca ffe ine and r em ai ned cha ng el ess i n ce lls treated wi th cy clo he xim ide．C y cli n E thresh—
old in J U R K A T cells w as much low er than that in M OI T 一 4 cells．It was sug g ested that F ormula
B ／ A × C w e fir stly set up co uld be used to ana ly ze cy cli n E e xpressio n thresho ld quantita tiv ely ．
K ey w o rd s：cycl in E ；th resh ol d ； q ua nti tativ e an aly sis
E expression at G 1／ S boundary is
w e tri ed to d ev elo p a qu an tita tive
a cha racter isti c fea ture o f cel l
a p pro ach to a na lyz e cyc li n E
E — t y pe cy clin s
ex pres sed du rin g t he lat e G 1 ph as e o f t he c ell c yc le
u nt i l t h e end o f t h e S— ph ase． T he act ivit y of c yc lin
E can limit th e p assag e of c ells th ro ug h t h e rest ric -
t ion p oin t “ R ” w h ich m ark s a “ po in t o f no ret ur n ”
f or cel ls en t erin g t h e div ision c yc le f rom a r est ing
s tat e o r pas sin g f rom G 1 int o S — ph ase
human tumo rs ．the exp ress ion levels of cyclin E is
s ignificant ly hig her than p hysio log ical ones．
Cyclins／ D NA muh iparameter flow cytometry
det ec t s b ot h ex pres sion of c yc lins and c el lu lar D N A
c on t en t ，w hic h mak e it po ss ib le t o relat e ex p res—
s ion o f eac h o f t h es e p ro tein s w ith t h e ac t ual cel l
cyc le po sit ion E 引
．
I n c on t ras t to ot h er c yc lins
cy c lin A ， B 1 ， D ) ， c yc lin E has a w ell def ined
thr eshold at G 1／ S boundaryE引．
s tu d y ，w e inv est igat ed t h e exp res sion of cy c lin E in
M 0 L T 一 4 cells under different photo muh ip lier tube
( P M T ) vo ltag es ，and in cells treated w ith different
d ru gs u n der th e s am e P M T v olt age． T he ex pres —
sion of cyclin E in M O I T 一 4 cM ls and J U R K A T
c ells w ere a lso co mp ared．
thres hold co uld b e q uantified by for mula B ／ A C
( A ，B ，C indicate the minimum ， threshold ，and
max im u m o f c yc lin E f luo resc enc e in t ens ity ， re—
spectively ) ．
( eyelin E 1 an d cy clin E 2 )are
． I n man y
( eg．
In the present
W e fo u nd c yc lin E
X IE D a xing ，m ale ，born in 19 80 ，M ． D ．，Ph． D ．
△ C orr espondinz author
T his project was supported by grants from the National
N a tur aI S ciences F ounda tion of C hina
3 9 730 2 70 ，and 39 72 50 2 7 ) ，the C hina S tate K ey Basic R e—
sear ch P rog r am( N o ． G 19 98 0 5 12 12 ) ， and the S cience
F o undatio n of M inistr y of H eah h。 P ． R ． C hina
20 0 125 3 7) ．
( N o． 39 67 20 65 ，
( N o ．
1 M A T E R I A L S A N D M E T H Do S
1． 1 C ell C ult ure an d T r eatm en t
T he M O L T 一 4 and J U R K A T cell 1ines w ere
pu rc ha sed fr om A meric an T y pe C u h u re C ol lec tio n
( R o ck v ille ，U S A ) ． T w o c elI lin es w ere c u lt u red in
R P M I 164 0 mediu m s u pp lemen t ed w it h 10
bovine ser um ， 100 U ／ ml penicillin 。 100 tLg／ ml
streptomy cin and 2 mmol／ I
media ，s u pp lem ent s ，an d sera w ere o bt ained f rom
G i bc o ( G rand I s land ，U S A ) ． T he c ells w ere main —
tained at the concer tration of 2 × 10 ／ ml in asyn—
ch ro no u s ex po nen t ial g row t h ． C aff ein e
U SA ) ，dis so lved in D M S O at a c on c en t rat io n of 5
mol／ I w as stored at 一20 。 C 。and a solution of cy —
clo h eximid e ( S ig ma ，U S A ) p repared b ef o re ex per—
imen t ． W hen t h ey w ere add ed t o c ells ， t h e f in al
co n cert rat ion o f c af f ein e an d cy clo he ximid e in t h e
media w ere 5 m mol／ I and 5 pmol／ I ，respectiv ely ．
1 ．2 I m m un o c y to c h em ist r y
T h e c e lls w er e h ar ve st e d f o r t h e ti m e m an —
tio n e d a b ov e ， w as h ed w it h P B S a n d t h en f i xe d
in s us p en sio n in 8 0 ethano l a t 一 2 0 。 C fo r a t
leas t 2 h o r o v er n ig h t． T h e s am P les w er e t h e n
c en t r if ug ed ， w as h e d w it h P B S ， an d
w it h 0 ．2 5 t rit o n X 一 1 0 0 f o r 5 m in o n ic e．
A f t er w a sh ed w it h P B S a n d c en t rif u g at io n 。 t h e
ceIls w er e incub ated a t 4
p r es en c e o f m o u s e m o n o c lo n al an t ib od y t o h u —
m a n c y c lin E ( P h a rm in g en ， U S A ) ， w h ic h w a s
d il u t ed 1 ：2 O 0 in P B S c on t ain in g 1
C eI ls w er e t h an w as h e d an d in c u b a t ed w it h a
F I T C— conjugated goat an ti— m ouse IgG antibody
f et al
I． -gl utamine． A ll
( S igm a ，
t reat ed
℃o v er nig ht in t he
B S A ．
报
学
学
g ∞大
m技
H
． 虽中
华
J

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X I E D ax ing et nZ．C y clin E T hreshold by C yclin E ／ D N A M uhiparameter F low C y tometry 28 3
( D A K 0 ， D e n m a rk )
t ai n in g 1
1 0 ug／ ml of P I and 0 ．1
an d in c u ba t ed a t ro o m t er n P e rat u re f o r 2 0 m in
p rio r t o m ea s u rem en t ． T h e c o n t ro l w a s p re—
p ar ed id en t ic al ly as d es c r ib ed
t h at t h e is o t y p e— s p ec if ic an t ib o d y
S igm a ，U S A )w as used ins tead of the cyclin E ．
C ellu lar f lu ores cen ce w as m ea su red u sin g th e F A C —
S o rt flow cytom tery( B ecton D ickinso n ， U S A ) ．
R ed and g reen emis sio ns f ro m eac h c ell w ere s ep a—
rated an d qu an tit at ed us in g t h e s t an d ard op t ics o f
t he F A C S o rt ． A ll t h e isom et ric c on t ou r m ap s o f
C yclin E ／ D N A in this study w ere analyz ed using
t he same c riterio n b y C ellq ues t s of tw are．
d ilu ted 1 ：2 0 in P B S c o n —
B S A fo r 3 0 m in ， re su sp en d ed in
R n ase A in P BS ，
ab ov e ，
( mou se IgG 1；
ex c ep t
2 R E SU L T S
2． 1 Quantitative A nalysis
F ig． 1 p resen t ed t he
cy clin E and D N A content in exp onentially g ro w ing
M o L T 一 4 c ells ．T h e e xp ress io n of c yc lin E w as c ell
cy cle sp ecific ，w hich reached maxim al in late G 1
and early S phas e cell s ( cell s in the rectang le ，fig ．
1 A ) 。t h en pro g res siv el y d ecl in ed d urin g S ph ase．
C ells in G ，and M phase w er e cyclin E neg ativ e． A
threshold of cyclin E expression at the G 1／ S
b ound ary w as ap par ent ( fig ． 1 A ； b lack ar row ；
fig． 1 B ) ． T he standard cyclin E thres hold w as cal—
culated by formula B ／ A × C ．
of C y c lin E T h resho ld
biv aria te dis t rIb ut ion of
0 20 0 4 00 60 0 80 0 l 000
D N A
B
C
_
b
a
0 200 4 0 0 6 00 800 l 0 00
D N A
F ig． 1 Isometric con tour m aps of C y clin E ／ D NA in
the exponential l y g ro w ing M 0 L T 一 4 cel ls
A ：B lack ar ro w sho w s the threshol d o f cyc—
l in E fl uo re scence intensity ．C el ls in the rec —
tang l e ( R ) w ere G l la te cell s ；
B ：T he m inim um ．thr esho ld ，and m ax im um
o f cy cli n E fluor escence intensity ( a ．b and
c ) wer e sho we d r especti vel y
2 ． 2
ferent P M T V olt ages
F ig． 2 presen t ed t h e ex pres sion of cy cl in E in
M o L T 一 4 cells und er d ifferent
( P M T ) v oltages ．T hresho ld w as calcula ted b y for—
m ula ( ta ble 1 ) ．W hen the ceIls w ere de tected un—
d er vario us P M T vo ltag es ，c yc lin E t hr es h old re—
mained in variab le ( fro m 2． 6 0 t o 2． 7 4) ．
T h reshold of C y c lin E E x p ression un der D if -
pho to m uh ip lier
Table 1 Q uantitative analysis of cyclin E threshold at differ。
en t P M T vol tages．A t d iff er ent P M T vol tages ，cy cli n
E threshold calculated by formula did not cha里
0 20 0 4 00 6 00 0 200 40 0 60 0 0 20 0 4 00 600
F L 3． a r e a F L 3． a re a F L 3 ． a re a
F ig． 2 Isom etric con tour maps of C y clin E ／ DN A in
M ( )I T 一 4 cell s under di fferent P M T vo ltag es
2． 3
clin E T h reshold in C ells T reated w it h D r ugs
F ig ． 3 p resen t ed c yc lin E e xp ress ion aft er
M o I T 一 4 cel ls w ere treated w ith caffeine and cyclo—
h ex im ide ( C H X )un d er th e s ame P M T set t in g s．
C y c lin E t h resh o ld w as d ec reas ed in c ells t reat ed
w it h caf fein e ( f ro m 2．10 to 0 ． 49 ，t ab le 2)
no t c h ang e in t h os e t reat ed w it h c yc loh exim id e
( f rom 2 ． 1O t o 2 ． 10 ，t ab le 2 ) ．
U n der t he Sam e P M T V olt age，C h anges of C y 。
and did
0 2 00 40 0 60 0
F L 3 - a rea
0 20 0 4 00 6 00
F L 3． ar ea
0 2 00 40 0 60 0
F L 3 - a rea
F ig． 3 Isometric contour maps of Cy clin E ／ D N A in
M O L T 一 4 cel ls tre ated w ith different dr ug s
A ：U ntrea ted cel ls ( contro 1) ；B ：C ell s tr ea —
ted w ith 5 mmol／ I caffeine；C ：C ells treated
w ith 5 tlmol／ I cyclohex imide
2．4
Sam e P M T V ol tages
F ig ．4 show ed the e xp ress ion levels of cycl in
E in J U R K A T cells w ere much Iow er than in
C y c lin E t hr esh old o f D if f er en t C ells u nd er th e
00 0 _ 【 00 ∞ 0 0 00 寸 00 0
三 ∞ }{ ． _ 【 厂 I
00 0 _ 【 00 ∞ 00 0 0
三 ∞ }{ - I
00 0 —0 0∞ 0 0 00寸 0 0 0
uII u∞ ． IoIl IIIlu U

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284 J ournal of H uazhong University of Science and T echnology [Med Sci] 25 (3) ： 282 284， 2005
M O I T 一 4 cells ( tab le 3 ) ．
T able 2 Q uantitative analysis of cyclin E threshold in
cell s t reated w i th d if fe rent d r ugs． C al cul ated
b y fo rm u la ，C ycl i n E th resho ld w as decr eased
obv ious ly in M ol t-4 c el ls tr ea ted w it h c aff ei n e
( 5 mmoI／ L ) and did not change in those with
cyclohex imide ( CH X ，5 p,mol／ L )
F i g． 4
0 2 00 40 0 6 00 800 l 0 00
D N A
0 20 0 4 00 60 0 80 0 l 00 0
D N A
Isometric contour maps of cyclin E ／ D NA in
M0 L T 一 4 cells and J U R K A T cells under the
sam e P M T v o ltag e
A ：M 0 L T 一 4 cells ；B ：J U R K A T cells
c enc e of la te G 1 p has e cell s ， resp ect ively ． I f t h e
P M T v olt ag es w ere inc reased ， th e sig nals o f “ B ”
a nd “ C ”w ere amp lif ied ． es pec ia lly th e s ign al o f
“ B ’’ w hic h w as m or e s en sit iv e t han t ha t o f “ C ” ．B y
co nt ras t ．t h e sig nal of “ A ”
G ph as e cells d idn't ex pres s cy c lin E ．A f t er s taf f s—
tic al a nd m at hem atic al an aly sis 。w e co nc lu d ed t h at
fo rm ula B ／ A× C co uld ex clud e the affectio n of
P M T vo lt age set t ing s．
d eg radat ion of c yc lin
t h resh o ld calc u lated b y
b le．
C affeine can ab olis h
thro ug h inh ib iting the activity o f A T M k inas e ，and
b y w h ic h in c reas e t he c el l i rradiat io n sen sit ivi—
t y E。 ‘
．
H o w eve r ， it s ef fec t on cy cli n E t h resh o ld
has ra rely b een s tud ied ．O ur s tud y s ug g es ted that
c aff eine c ou ld dec rease c y clin E th resh o ld a t t he
concentration reco mm end ed．
vate cyclinE ／ CD K 2 kinase activity ，hence acceler—
at e c ells go ing th ro ug h G l an d S p h ase ，and s ho r t—
en the d ura tio n of G 1 p has e．
C yc loh ex imide 。 a p ro t ein
t o r L
，can res train ot h er G 1 o r S c yc lin s ( eg ．c yc lin
D and cyclin A ) and inhibit G n and G 1／ S phase pro—
gression．F or the reason that the duration of G 1／
G o ph ase pro lon ged ，c yc lin E ac cu m ula ted grad ual —
ly an d r eac hed t h res ho ld at no rma l pro t ein levels
u ltim ate ly．
T h resh o ld of cy clin E m ay dif f er in vario us
cell ty pes ． O ur analy sis ind icated that cyclin E
threshold in J U R K A T cel ls was m uch low er than
t hat o f M O L T 一 4 c ells ， w h ich is p rob ab ly d u e to
t he dif fer en ce in t h e s peed o f p ro tein s yn t h es is a nd
t he du rat io n o f G 】ph as e．
ch an ged lit t le bec au se
S ince the sy nthesis a nd
E w ere n ot af f ec t ed 。 its
fo r mula s ho uld b e invaria—
cel l cycle check point
C affeine mig ht acti—
s yn t h es is in h ibi—
Table 3 Quantitative analysis of cyclin E expression thre s hold
in d if fer ent c el l typ e s
R EF ER E NC E S
3 D I SC U SS10 N
U sing C y clin E ／ D N A multiparameter flow cy —
t om etr y ，G o n g h ad s h ow ed th at a t h res ho ld of cy c —
lin E expression at the G 1／ S boundary is apparent ；
an d t h e ce lls belo w th is th res ho ld ( nam ed as ear ly
G 1 ce lls ) d o n o t en ter S ph ase[0’
c yc lin E f luo resc enc e c h ang ed w hen w e d etec ted
cells und er d ifferent P M T vo ltages 。how ever 。it
should be invariab le in nature． T o av oid the dis —
turb ance o f d ifferent P M T vo ltag es ，w e set up a
new for mula o f B ／ A ×C to d eterm ine cy clin E
th res ho ld qu an tit at ively． H o w did w e c on c lud e t his
for mu la? T h e th resh o ld o f c yc lin is rela ted to it s
synthesis and deg radatio n s p eed ．and iS also relat—
ed to it s d u ratio n i n th e c yc le． O n t h e is om etr ic
contour maps of C y clin E ／ D N A 。“ A ” 。“ B ”
“ C ” in dicat e t h e min imal f l uo rescen c e o f G p h ase
cells ．1ate G 1 phas e cells and maxim um fl uo res 一
．
I n th is met ho d ，
and
1 M oroy T ，Geisen C ．C y clin E ．Int J B iochem C ell B iol，
2 00 4 ， 3 6 ： 1 42 4
G ong J ，L i X ，T raganos F el a1．E x pression of G 1 and
G 2 cycl ins m ea sur ed i n i ndiv idua l cel ls by m uh iparam e te r
flo w cyto m etry ：A new to o h n the a nal y si s o f the cel l cy—
c le．C e ll P ro l if ，19 94 ， 2 7 ： 3 57
Gong J ，T rag anos F ，Darzy nk iew icz Z．Discrimination of
G 2 and m i to ti c cei ls by f low cy to m et ry based o n dif fe rent
expr essio n of cycl ins A and B 1． E xp C e Il R e s。 19 95 ．
22 0 ： 2 26
G ong J ，T rag anos F ，Darzynk iew icz Z．G row th imbal—
a nce and a lte red e xpre ssio n of cy cli ns B 1 。A ．E ．a nd D 3
in M O L T 一 4 ceil s synchro ni zed in the cel l c ycle by i nhibi—
tor s o f D N A repli catio n． C ell G ro wth D iffe r ， 1 9 9 5 ， 6 ：
1 48 5
B lasi na A ，P rice B D 。T urenne G A el a 1．C affiene inhib—
its the checkpo int k inase A T M ． C ur r B io l ，19 99 。 9 ： 11 35
M oser B A ，B rondello J M ， Baber- F urnari B et a1．
M echanism o f caffeine- i nduced check po int o ve rr ide in f is—
sion y east． Mol C ell Biol ，2 00 0 ， 2 O： 42 8 8
S ak ai T ，A o ik e A ， M a r ui N el a 1． P ro tectio n by cy clo —
he xi m ide aga inst cyto tox ici ty induced by v incri stine ，co l —
chicine．or delta 12-prostag landin J 2 on human osteosar—
co m a cei ls． C ancer R es ，19 89 ， 4 9 ： 1 19 3
( R eceiv ed F eb．2 8 。2 00 5 )
2
3
4
5
6
7
0 00 _ l 00∞ 0 0 00 寸 0 0 0
ou1 Iou啦 o． 1 0j 岛 1 ll l u U