Download —(5),weexaminedwhethertheadditionofMVA

INCADRONATE
INHIBITSThE MVA PATHWAYIN MYELOMACELLS
Results
A
Incadronate-induced
300'
Apoptosis in Myeloma Cells Can Be Pre
vented by FOil and GGOH. In accord with our previous study (5), we
found that 100 @iM
incadronate caused an increase of approximately
-@-
250 -
150% in the proportion of morphologically apoptotic JJN-3 myeloma
cells after treatment for 72 h (Fig. 1A). This effect was prevented by the
addition of 20 @J@M
FOH or GGOH. FOH and GGOH are analogues of
-a 200'
FPPandG@IPP,
respectively,
withagreater
membrane
permeability
than
0
LI
the W1 forms (1 1). The addition of 1 mi@iMVA had no effect on
incadronate-induced apoptosis. Similarly, using a fluorescence in situ
nick translation assay, (which we have previously used to detect DNA
strand breaks in JJN-3 myeloma cells; Ref. 5), 20 @u@i
FOH or GGOH
prevented incadronate-induced apoptosis, whereas 500 @u@i
MVA had a
@aI
protective effect (TaMe 1). On the basis of [31-flthymidine incor
poration, 10 @M
FOH or GGOH or 1 mrs MVA had no significant effect
on the inhibition of cell proliferation induced by incadronate (Fig. 1B).
,@
a
l50@
a
n@100.
*
50 I
0@ I
I
JJN.3
Myeloma
Cells. Recent
studies
•
INC
MevastatinCausesApoptosisand InhibitsProliferationof
with J774 macrophages
have shown that mevastatin is a potent inducer of macrophage
INC+
MVA
INC+
FOH
INC+
GGOH
B
apoptosis, and that this effect can be partially prevented by the
addition of intermediates of the MVA pathway, such as FPP and
125
GGPP (9). In the present study, mevastatin treatment of JJN-3
myeloma cells for 72 h also resulted in an increase in the propor
100
tion of apoptotic cells from 15.1 ±0.5% in control cultures to
542 ±2.4 and 88.8 ±1.7% (P < 0.05 compared to control) in
cultures treated with 10 and 50 @LM
mevastatin, respectively. Ap
optotic cells were identified on the basis of changes in
morphology characterized by chromatin condensation and
fragmentation (5). Treatment for 72 h with mevastatin also
in a dose-dependent inhibition of proliferation. Treatment
a
0
LI
nuclear
nuclear
resulted
with 10
75
0
a
U
and 50 @LM
mevastatin caused a 67.5 ±2.6 and 80.9 ±2.3%
reduction (compared to control; P < 0.01) in the incorporation of
[3H]thymidine into JJN-3 myeloma cells.
Induction of Apoptosis and Inhibition of Proliferation by Me
50 -
25-
0-
vastatinin MyelomaCellsCanBePreventedby MVAor GGOH.
uiil
I
Ctl
I
I
I
INC
INC+
INC+
INC+
MVA
FOH
GGOH
Using changes in nuclear morphology as an indication of apoptosis,
I
the additionof 1 mr@i
MVA or 20 @M
(X3OHpreventedmevastatin
Fig. 1. Effect of the MVA pathway intermediates MVA, FOH, and GOGH on (A)
induced apoptosis (Fig. 2A). However, 20 ,.LMFOH did not signifi
candy prevent apoptosis. These observations were confirmed using a
fluorescence in situ nick translation assay (Table 1). The addition of
1 mM MVA or 10 @LM
GGOH also prevented the inhibition of prolif
incadronate (iNC)-induced apoptosis (R) and (B) incadronate-induced inhibition of pro
liferation (D). JJN-3 myeloma cells were treated with 100 @si
incadronate in the presence
or absence of MVA, FOH, or GGOH. Results are expressed as a percentage of the
appropriate control (control, 0). Data are the mean ±SE (A, n = 4; B, n = 6) of a
representative experiment. a, p < 0.05 compared to incadronate alone.
eration
caused
by 30
@M
mevastatin
(Fig. 2B). The addition
of 10
@M
FOHhadnosignificant
effectontheinhibition
ofproliferation
caused
by mevastatin.
FOHand GGOHHavePartiallyProtectiveEffectson Cell
Cycle Arrest Induced by Incadronate. Givenour previousobserva
tions that incadronate causes both apoptosis and cell cycle arrest in S
Discussion
Bisphosphonates are a class of drugs that target bone mineral
and inhibit the function of bone-resorbing
osteoclasts (1). Re
cently, breakthroughs have been made in understanding the mo
lecular mechanisms that may be involved in the events that lead to
osteoclast apoptosis. Following the report by Amin et a!. (12) that
nitrogen-containing
bisphosphonates can inhibit sterol biosynthe
—(5),weexamined
whether
the
addition
ofMVA,
FOH,
orGGOH
could prevent these effects on the cell cycle. Flow cytometric analysis of
propidium iodide-stained JJN-3 myeloma cells showed that treatment
with 100 @LM
incadronate increased the proportion of hypodiploid apop
totic cells (sub-G@,/G1phase) and caused an increase in the proportion of
cells in S
@se(Fig. 3). The increase in the proportion of hypodiploid
apo@c cells was prevented by the addition of 20 piti FOH or GGOH,
sis in macrophages,
we found
that inhibition
of the same
biosyn
thetic pathway (the MVA pathway) by these bisphosphonates
causes macrophage apoptosis. However, apoptosis in macrophages
is probably
the consequence
of a loss
of isoprenylated
with the hypodiploid population reduced to control levels, whereas 500
proteins
rather than a loss of sterols (9).
@JMMVA had a partial protective effect. The effect of incadronate on the
In addition to affecting osteoclasts, bisphosphonates have been shown
cell cycle could be partially overcome by the addition ofFOH or GGOH,
to have effects on tumor cells in vitro, including the induction of apop
but not by the addition of MVA. As expected, the addition of 500 gii@i tosis in human myeloma cell lines (5) and in plasma cells isolated from
MVA or 20 pi@tGGOH but not 20 pi@iFOH completely prevented the patients with multiple myeloma (6) and the inhibition of adhesion of
effects of mevastatin on the cell cycle (Fig. 3).
breast and prostate cancer cells to mineralized and unmineralized bone
5295
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INCADRONATE INHIBITS ThE MVA PATHWAY IN MYELOMA CELLS
Table I incadronate- and mevastatin-induced apoptosis in JJN-3 cells is prevented by
the addition of intermediates
pathwayApoptosis
of the MVA
control)@'Treatment'@
MevastatinNone
(% of
100 p.MIncadronate
500@MMVA
20 @M
FOH
[email protected]
a JJN-3
@
cells
were
cultured
with
100
267
168
120
93
100
223
102
@LMincadronate
or absence of 500 gasiMVA, 20 @zi@t
FOH, or 20
treated with either MVA, FOH, or 000H
20 p.st
219
or 20 pi@i mevastatin
in the
presence
000H for 72 h. Control cultures were
in the absence of incadronale or mevastatin.
b Apoptosis was measured using a fluorescence in situ nick translation assay and analyzed
by flow cytometiy. Results are expressed as a percentageof the appropriatecontrol.
matrices (13, 14). In vivo, bisphosphonate treatment has been shown to
inhibit the progression and development of bone metastases and reduce
the tumor burden in a mouse model of breast cancer (15), and, in some
instances, bisphosphonate treatment may prolong the survival of patients
with multiple myeloma (3, 4). Bisphosphonale treatment has also recently
been demonstrated to increase survival in patients with breast cancer(l6).
These observations raise the possibility that bisphosphonates may have
antitumor effects (either direct or indirect) in addition to their antiresorp
tive effects on osteoclasts (17, 18).
MVA or GGOH but not FOH. The addition of either MVA or
GGOH also prevented mevastatin-induced changes in the cell
cycle. This is consistent with other reports that geranylgeranylated
proteins, rather than farnesylated proteins, are required for cell
cycle progression (19). Similar to incadronate-induced
apoptosis,
the effect of incadronate on the cell cycle could be partially
overcome by the addition of FOH or GGOH but not MVA. Neither
FOH, GQOH, nor MVA prevented the inhibition of proliferation
induced by incadronate (by measuring [3H]thymidine incorpora
tion). However, this may be a consequence of the lower concen
trations of FOH and GGOH that had to be used in this assay, due
to the ability of FOH and GGOH alone to inhibit cell proliferation
(data not shown). These observations suggest that the antiprolif
erative effects as well as the apoptosis-inducing
effects of incad
ronate may be the result of a loss of isoprenylated proteins.
Statins such as lovastatin are known to cause apoptosis in prostate
tumor cells in vitro (20). Other inhibitors ofprotein prenylation have been
intensively studied as potential antitumor agents that may specifically
A
500 I
Inthisstudy,we havedemonstrated
thatthenimtgen-contaimng
bisphos
@nate
incadrOnate causes the apoptosis of a human myeloma cell line in
vitm by inhibiting the MVA pathway. Mevastatin, an inhibitor of HMG-CoA
400!
reductase (the rate-limiting enzyme in the MVA pathway that catalyzes the
synthesis of MVA), is even more potent than incadronate in causing my
eloma cell apoptosis.
It is likely that apoptosis
induced by either of these
0
agents is a consequence ofthe loss of famesylated and/or geranylgeranylated
proteins. The addition of MVA (which can be metabolized to isopentenyl
PPi, FPP, and GGPP) directly bypassed the site of inhibition of mevastatin
and completely prevented apoptosis. However, FOH did not prevent
@300
0
LI
0
a
U
2
mevastatin-induced apoptosis. Although FOH is metabolized to FPP, cuntnt
evidence suggests that little of this FPP is metabolized further to GGPP (1 1),
200
100
and FOH is therefore only used for protein famesylation. In addition, the
convemionofFPP to GGPP requiresisopentenylPP1.the synthesisof which
@
@
(like FPP and GGPP) is indirectly prevented by mevastalin. Hence, it is
unlikely that a substantial amount ofFOH can be convefled to GGPP via FPP
in the presence of mevastatin. The @kIiflon
of GGOH alone (which can be
metabolized to GGPP, the substmte required for geranylgeranylation of
proteins; Ref. 11) was sufficient to prevent mevastatin-induced apoptosis.
Together, these observations suggest that gemnylgeranylated proteins, rather
than famesylated proteins, are the dominant forms of isoprenylated proteins
required for the suppression of apoptosis in JJN-3 myeloma cells.
The addition of GGOH prevented incadronate-induced apoptosis,
probably by replenishing the intracellular pool of GGPP required for
protein isoprenylation. Apoptosis induced by incadronate could not be
prevented by the addition ofMVA, because incadronate appears to inhibit
an enzyme(s) in the MVA pathway required for the synthesis of FPP or
GGPP from MVA (9). These observations are consistent with the above
hypothesis that gemanylgeranylated proteins prevent apoptosis in JJN-3
cells. However, FOH also prevented incadronate-induced apoptosis. Al
though it has been suggested that FOH cannot be metabolized to GGPP
(11), as discussed above, it is possible that in the presence of incadronate
0—
I
Mev
fl
I
U
r@-i
I
Mev +
MVA
Mev +
FOH
Mev +
GGOH
Mev +
MVA
Mev +
FOH
Mev +
GGOH
B
150
125
_100
a
0
LI
75
a
U
H
5f@.
25
(but not mevastatin),a small amount of FOH may be converted to GGPP
via FPP, which is sufficient to temporarily rescue the cells from apopto
sis. Alternatively, JJN-3 cells may convert FOH to FPP for the farnesy
lation of proteins that are normally geranylgeranylated (10).
We and others have shown that in addition to causing apoptosis,
bisphosphonates can cause an accumulation of cells in S phase of
the cell cycle and inhibit cell proliferation (5, 6). In this study, we
found that mevastatin also inhibited the proliferation of JJN-3
myeloma cells, an effect that could be overcome by the addition of
0@
CU
Mev
Fig. 2. Effect of the MVA pathway intermediates MVA, FOH, and 000H
on (A)
mevastatin (Mev)-induced apoptosis (@) and (B) mevastatin-induced inhibition of prohferalion
(D). JJN-3 myeloma cells were treatedwith 30 @zs.i
mevastatin in the presenceor absenceof
MVA, FOH, tw 000H. Results are expressed as a percentage of the appropriate control
(control.0). Data are the mean ±SE(A. n = 4; B, n = 6) ofa representativeexperiment *.
P < 0.05; @*,
P < 0.01 comparedto mevastatinalone.
5296
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
INCADRONATE INHIBITS THE MVA PATHWAY IN MYELOMA CELLS
40
GWG1
40'
(a)
30
a
n
0
LI
10
U
I
200
400
20
20
10
10
I
@
content ofJJN-3 myeloma cells after labeling with
30propidium iodide. Cells were cultured with: a, PBS;
b, 100 @AM
incadronate;c, 20 pat mevastatin; d,
a
n
incadronate + 500 @zat
MVA; e, incadronate +20
0
LI
@&M
FOH;f, incadronate + 20 pat GGOH; g, mev
astatin + 500 @LM
MVA; h, mevastatin + 20 @si
10@
FOH; and I, mevastatin+ 20 pat GGOH. Results
â€ãre
from
arepresentative
experiment,
and
the
pop
@
@
ulations of hypodiploid (H) cells or cells in the
DNA content
(0
(e)
30
30.
20
20.
10
10
2O0
2@4ó0600800
G0k31 phase, S phase. and G2-M phase of the cell
200400600800
40.
40
(d)
Fig. 3. Flow cytometricanalysisof the DNA
I
200400600800
DNA content
DNA content
40-
(c)
30
800
600
(b)
30
@tOo 6O0
)
8O0
200
400
600
800
DNA content
DNA content
DNA content
cycleareindicated.
40
40
(g)
30
(h)
(i)
30
30
20
20
10
10
a
0
40
LI
200
400
600
800
0
0
200400600800
DNA content
DNA content
J
200 400 6óo 800
DNA content
8. Faith, J. C., Mönkkdnen,J., Blackburn, G. M., Russell, R. G. G., and Rogers, M. J.
Clodronate and liposome-encapsulated clodronate are metabolized to a toxic AlP
analog, adenosine 5'-(@,y-dichloromethylene) triphosphate, by mammalian cells in
disruptthe function ofisoprenylated Ran proteins (reviewed by Gibbs and
011ff; Ref. 10). We have demonstrated that bisphosphonates, a class of
antiresorptive drugs, can cause human myeloma cell apoptosis in vitro by
inhibiting the pathway required for protein isoprenylation. It remains to
be determined whether sufficient concentrations of bisphosphonate may
be generated in the bone microenvironment in vivo to have a direct
apoptosis-inducing effect on tumor cells as well as on osteoclasts. How
ever, our observations that some bisphosphonates can cause apoptosis in
10. Gibbs, J. B., and Oliff, A. The potential of farnesyltransferase inhibitors as cancer
chemotherapeutics. Annu. Rev. Pharmacol. Toxicol., 37: 143—166,
1997.
11. Crick, D. C., Andres, D. A., and wa@chter,C. J. Novel salvage pathway utilizing
myeloma cells (and other cell types in vitro; Ref. 9) by interfering with a
farnesoland geranylgeraniolfor proteinisoprenylation.Biochem.Biophys.Rca.
metabolic pathway required for the function of Ras highlights the ques
tion of whether bisphosphonates might have direct antitumor effects in
vivoin diseasessuchas multiplemyeloma.
References
1. Fleisch. H. Bisphosphonates:pharmacologyand use in the treatmentof tumour
induced hypercalcaentic and metastatic bone disease. Drugs, 42: 919—944,1991.
2. Jantunen, E., and Laakso, M. Bisphosphonates in multiple myeloma: current status;
future perspectives. Br. J. Haematol., 93: 501—506,1996.
3. Berenson,
J.R., Lichtenstein,
A., Porter,L, Dimopoulos,
M. A., Bordoni,R.,George.
S., Upton. A., Keller, A., Ballester, 0., Kovacs, M., Blacklock, H., Bell, R., Simeone,
vitro.
J.Bone Miner.Res.,12: 1358—1367,
1997.
9. Luckman, S. P., Hughes, D. E., Coxon, F. P., Russell, R. G. G., and Rogers. M. J.
Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent
pest-translational prenylation of GTP-binding proteins. including Ras. J. Bone Miner.
Res.,13: 581—589,
1998.
Commun., 237: 483—487,1997.
12. Amin, D., Cornell, S. A., Gustafson, S. K., Needle, S. J., Ullrich, J. W., Bilder,
G. E., and Perrone, M. H. Bisphosphonates
used for the treatment of bone
disorders inhibit squalene synthase and cholesterol biosynthesis. J. Lipid Res., 33:
1657—1663,
1992.
13. Van dci Pluijm, G., Vlocdgraven, H., Van Beck, E., van der Wee-Pals, L., Lowik, C..
and Papapoulos, S. Bisphosphonates inhibit the adhesion of breast cancer cells to
bone matrices in vitro. J. Clin. Investig., 98: 698-705, 1996.
14. Boissia,S., MagnettO,S.,FrappaII@L,OIZin,B.,EbetinO,
F. H.,DC1maS,P.
D.,andCleZadin,
P. Bisphospinnates inithit prostateand breastcarcinomacell adhesion to immineralizedsad
mineralizedbone extrseellularmatrices.CaneerRca.,57: 3890-3894, 1997.
15. Sasaki, A., Boyce, B. F., Story, B., Wright, K. R., Chapman, M., Boyce, R.,
Mundy,G. R., and Yoneda,T. Bisphosphonaterisedronatereduces metastatic
human breast cancer burden in bone in nude mice. Cancer Res., 55: 355 1—3557,
1995.
J. F., Reitsma, D. J., Heffernan, M., Seaman, J., and Knight, R. D. Long-term
pamidronate treatment of advanced multiple myeloma patients reduces skeletal
16. Did, I. J., Solomayer, E-F., Costa, S. D., Gollan, C., Goemer, R., Wallwiener, D.,
events. J. Cliii. OncoL, 16: 593—602, 1998.
adjuvant clodronate treatment. N. Engl. 3. Med., 339: 357—363,1998.
17. Mundy, G. R., and Yoneda, T. Bisphosphonates as anticancerdrugs. N. Engl. J. Med.,
4. McCloskey,E. V., MacLennan,I. C. M., Drayson,M. T., Chapman,C., Dunn,3., and
Kanis, J. A. A randomized trial of the effect of clodronate on skeletal morbidity in
multiple mycloma. Br. J. Haematol., 100: 317—325,1998.
5. Shipman, C. M., Rogers, M. J., Apperley, J. F., Russell, R. G. G., and Croucher, P. I.
Biaphosphonates induce apoptosis in human myeloma cell lines: a novel anti-tumour
a@ty. Br. 3. HaematoL, 98: 665-672. 1997.
Kaufmann, M., and Bastert, G. Reduction in new metastases in breast cancer with
339: 398—400,1998.
18. Shipman. C. M., Rogers, M. J., Apperley, J. F., Russell, R. G. G., and Croucher, P. I.
Anti-tumoureffect
ofbisphosphonates
in human myeloma cells. Leuk. Lymphoma, in
press, 1998.
19. Vogt, A., Qian, Y., McGuire, T. F., Hamilton, A. D., and Sebti, S. M. Protein
6. Aparicio, A., Gardner, A., Tu, Y., Savage, A., Berenson, J., and Lichtenstein, A. In
s'itm cytoreductive effects on multiple myeloma cells induced by bisphosphonates.
geranylgeranylation, not farnesylation, is required for the G1 to S phase transition in
Leukemia (Baltimore), 12: 220—229,1998.
7. Rogers, M. J., Watts, D. J., and Russell, R. G. G. Overview of bisphosphonates.
20. Marcelli, M., Cunningham, G. R., Haidacher, S. J., Padayatty, S. J., Sturgis, L.,
Cancer(Phila.),80: 1652—1660,
1997.
mouse fibroblasts. Oncogene, 13: 1991—1999,1996.
Kagan, C., and Denner, L. Caspase-7 is activated during lovastatin-induced apoptosis
of the prostate cancer cell line LNCaP. Cancer Res., 58: 76—83,1998.
5297
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CANCERRESEARCH58. 5298-5300. December1. 1998]
Advances
in Brief
Absence of Topoisomerase II@3in an Amsacrine-resistant
Line with Mutant Topoisomerase Ha1
Human Leukemia Cell
Cynthia E. Herzog,2 Katherine A. Holmes, Laura M. Tuschong, Ram Ganapathi, and Leonard A. ZweHing
University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [C. E. H., L M. T., L A. ii, and Cleveland Clinic, Cleveland, Ohio 44195 [K. A. H., R. G.]
Abstract
at least one mutation. Thus, these cells could provide an excellent
model in which to ascertain the relative contribution to drug sensitiv
ity of the a and (3 isoforms of topo H.
Numerous chemotherapeutic
agents act via stabilization
of a topoi
somerase (topo) Il-DNA complex. HL-60/AMSA, a human leukemia cell
line, is resistant to intercalator-mediated DNA complex formation and
cytotoxicity. HL-60/AMSA contains a mutant form of tape Ha that was
thought
to explain this resistance.
expression
oftopo
However,
II@ RNA in HL-60/AMSA
our present
Materials
data show that
is only 10% ofthat
and Methods
Cells. HL-60/AMSAcellswereobtainedfromDr.MiloslavBeranofM. D.
in HL-60,
Anderson Cancer Center and have been described previously
(1). The cells
and topo ll1@protein levelsare undetectable.Southern analysisoftopo II@3 were propagated in Iscove's modified Dulbecco's medium with 10% FCS at
shows no differences in gene dosage between the two cell lines but does
show differences in the restriction patterns. These data suggest that
decreased
topo II@J expression
may contribute
to the intercalator
37°Cin 5% CO2 with a doubling time of approximately 24 h. Cells were
periodically evaluated by American Type Culture Collection and found to be
resist
free of mycoplasma infection.
Whole Cell Assays. Soft agarcolony formationassays were performedas
ance of HL-60/AMSA cells.
described previously according to the method of Chu and Fisher (1). SDSIKC1
precipitation of DNA-protein complexes were performed as described previ
Introduction
ously (1).
Several chemotherapeutic agents exert their cytotoxicity via stabi
Northern and Southern Blots. RNAand DNAisolationfor Northernand
lization of a topo3 Il-DNA complex, which is thought to trigger
apoptotic cell death pathways. The mechanisms by which complex
stabilization and DNA strand breaks lead to cell death have not been
elucidated. However, in general the magnitude of drug-induced DNA
Southern blotting were performed using standard techniques. Probes for de
tection of topo 11f3were Fl2, which starts at bp 3915 of the coding region and
extends into the 3'-untranslated region (9) and SP12, extending from approx
imately bp 1000 to bp 3114 (Ref. 10; Fig. 1). Fl2 and SP12 were gifts from
Dr. Caroline Austin (The University of Newcastle-upon-Tyne, Newcastle,
strand
break
production
correlates
with
the
magnitude
of drug
induced cytotoxicity (1, 2). Two isoforms of topo II have been
identified, the a and f3 isoforms, whose genes are located on chro
mosomes l7q and 3p, respectively (3). Expression of the a isoform
varies during the cell cycle, whereas expression of the @3
isoform
United Kingdom) and Dr. K. B. Tan (SmithKline Beecham, King of Pnissia,
PA), respectively. Other probes were the human topo Ha probe, Z1169,
provided by Dr. Leroy Liu (University of Medicine and Dentistry of New
Jersey, Piscataway, NJ; Ref. 1) and the l800-bp PstI fragment of chicken
f3-actin cDNA.
changes little throughout the cell cycle (4, 5). Although topo II is a
Immunoblotting. Whole cell lysates from 106 cells were prepared in
critical enzyme for cell proliferation, the exact role of the two iso
2 X Laemmli
forms is unknown.
40% glycerol,
Two recent reports
have suggested
that amsacrine
may target the topo 11(3isoform. An amsacrine-resistant subline of the
human small cell lung cancer cell line GLC4 has levels of topo lla
comparable to the parental cell line, but the topo 11(3is only about 20%
of the parental level (6). Dereuddre, et a!. (7) reported increased
drug-induced topo II DNA cleavage and a 90% increase in cytotox
icity with amsacrine after transfection of topo 11(3into the Chinese
hamster lung cell line DC-3F/9-OH-E, selected for resistance to the
phenotype of HL-60/AMSA, a human leukemia cell line containing a
mutated
topo lIce (1 , 8). Our results
demonstrate
that these cells are
indeed deficient in both the transcript and protein for topo 11(3.We
also show that the gene for topo II@3is present in these cells but has
Received 9/3/98; accepted 10/16/98.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I Supported
2 To whom
by NIH Grants CA40900
(to L. A. Z.) and CA35531
(to R. G.).
requests for reprints should be addressed,
University
of Texas
M. D.
Anderson Cancer Center, 1515 Holcombe Boulevard, Pediatrics, Box 87, Houston, TX
77030.
Phone:
(713)
745-0157;
Fax:
(713)
mdacc.tmc.edu.
3 The
abbreviation
used
is: topo,
topoisomerase.
794-5531;
E-mail:
cherzog@notes.
1.4 M f3-mercaptoethanol]
by sonication
for 30 s, followed
blue,
by
boiling for 5 mm. Proteins were resolved on a 5% SDS-polyacrylamide gel,
electroblotted
selectively
onto nitrocellulose,
recognized
and probed with specific antisera which
topo Ha or 43 provided
by Dr. Ian Hickson
(Imperial
Cancer Research Fund Laboratory, Oxford, United Kingdom; Refs. 11, 12) or
topo I provided by Y. C. Cheng (Yale University School of Medicine, New
Haven, CF; Ref. 13).
Results
intercalator 9-OH-ellipticine. These results suggest a specific mech
anistic connection between amsacrine sensitivity and topo II@. The
human leukemia cell line, HL-60/AMSA, is remarkably more resist
ant to amsacrine than to etoposide (1). We, therefore, postulated that
altered expression of topo H@3may contribute to the unique resistance
buffer [125 nmi Tris (pH 6.8), 4% SDS, 0.25% bromphenol
Measurement
of Effects of Etoposide and Amsacrine on IlL
60/AMSA Cells. Measurement of cytotoxicity by soft agar colony
formation assays showed that HL-60/AMSA
cells were resistant to
amsacrine and, to a lesser degree, etoposide (Table 1). SDSIKC1
precipitation assays were performed to measure drug-stabilized topo
11-DNA complex formation. In accordance with the colony formation
assays, reduction in drug-induced topo Il-DNA complex formation in
HL-60/AMSA relative to HL-60 is greater with amsacrine than with
etoposide (Table 1). The results of both assays are also consistent with
previously published reports on the resistance phenotype of the HL
60/AMSA cells (1).
Northern Analysis of topo llfi in HL-60/AMSA. Studies done
with the F12 probe showed barely detectable expression of topo 11@3
RNA in the HL-60/AMSA cells, whereas a strong signal was seen in
HL-60 parent cells (Fig. 2A). Because a truncation mutation in topo
11@3
had been described previously (7), we reevaluated RNA expres
sion with probe SP12, which contains the middle portion of the
5298
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
Reversible, p16-mediated Cell Cycle Arrest as Protection from
Chemotherapy
Steven Stone, Priya Dayananth and Alexander Kamb
Cancer Res 1996;56:3199-3202.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.