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Blood, 1 September 2001, Vol. 98, No. 5, pp. 1630-1632
CORRESPONDENCE
To the editor:
CD30-activation-mediated growth inhibition of anaplastic
large-cell lymphoma cell lines: apoptosis or cell-cycle arrest?
We read with interest the Mir et al article on the
effects of CD30 activation on anaplastic large-cell lymphoma and
Hodgkin disease cell lines.1 The authors demonstrated
apoptotic responses and lack of an nuclear factor (NF)- B response,
which is presumed to be responsible for the apoptotic response,
following CD30 activation of the anaplastic large-cell lymphoma
cell lines. We performed numerous experiments on the cell lines mentioned in the
study. Activation of the CD30 signaling pathway by HeFi-1, a CD30
activating antibody,2 caused a significant decrease in
thymidine uptake of nodal ALCL cell line Karpas 299.3 But we did not observe apoptosis of the Karpas 299 cell line following CD30
activation measured by double staining with propidium iodide and
Annexin V (not shown). To determine the effects of CD30 activation on cell-cycle regulation,
we investigated the status of retinoblastoma protein and p21 following
CD30 activation by immunoblotting. Binding was detected using an
ImmunStar chemiluminescence detection system (BioRad, Hercules, CA).
There was an increase in the unphosphorylated retinoblastoma protein
(Rb) in CD30-activated Karpas 299 cells at 40 hours after CD30
activation compared with untreated (control) Karpas 299 cells that had
almost entirely phosphorylated Rb (Figure 1). Likewise, at 40 hours there was no
expression of p21 by the control cells while CD30 activated Karpas 299 cells had a high level of p21 expression (Figure 1).
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| Figure 1.
Expression of Rb protein and p21 in Karpas 299 cells
after CD30 activation.
After synchronization, cells were fed serum and allowed to grow. Forty
hours after addition of serum, in the absence of HeFi-1 all
the Rb protein expressed is in phosphorylated form (upper band),
whereas the HeFi-1 treated cells demonstrate a significant amount of
unphosphorylated Rb protein. p21 is not expressed in the control cells,
whereas the CD30-activated cells have high p21 expression at 40 hours
after CD30 activation.
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We also investigated gene expression patterns of the Karpas 299 cells before and after CD30 activation by use of the Atlas human
complementary DNA (cDNA) array for apoptosis (Clontech, Palo Alto, CA),
which contains 205 genes associated with apoptosis and cell-cycle
regulation. We found that upon CD30 activation most genes involved in
cell-cycle progression showed decreased expression. Most significantly,
proliferating cell nuclear antigen (PCNA), cyclins A, B, and H
were down-regulated, as were the genes for caspase 1, 2, 3, and 4 (not shown). Expression of the gene for antiapoptotic protein
IAP-1 was increased. To understand the role of NF-B in the growth inhibitory action of
CD30, NF-B activity was determined. Nuclear extracts from cell lines
before and after stimulation with HeFi-1 for 1 hour were obtained. The
extracts were incubated with 32P end-labeled NF-B
binding site oligos (5'-AGCTTGGGGTATTTCCAGCCG-3') and excess cold
oligos and run on a nondenaturing 6% PAGE (Gelshift kit, Geneka,
Montreal, QC, Canada). There was a high constitutive activity in
KMH2 while Karpas 299 had no activity. After incubation with
HeFi-1 for 1 hour, KMH2 had no change in activity (not shown), but
Karpas 299 cell line showed modest NF-B activation (Figure 2A). To further investigate the role of
NF-B on the CD30 action, we utilized a selective NF-B inhibitor,
SN50, which is a synthetic cell permeable peptide and inhibits NF-B
by binding to the nuclear localization sequence of the p50 subunit of
NF-B.4 The KMH2 cell line demonstrated a significant
decrease in 3H-thymidine uptake within 24 hours with 50 µg/mL of NF-B inhibitor SN50. Incubation with HeFi-1 combined with
SN50 had no significant further effect on the KMH2 response. Karpas 299 was not growth-inhibited by SN50 alone, while the combined treatment
with HeFi-1 and SN50 enhanced the inhibitory effect of HeFi-1 (Figure
2B). Our results differ from the data presented by Mir et al in 2 respects. We could not demonstrate apoptosis in response to CD30
activation on Karpas 299 cells, while we did observe NF-B activation
after CD30 activation. Additionally, we have evidence of a
cell-cycle-inhibitory effect of CD30 activation on the Karpas 299 cell
line. The discrepancies between our results and theirs could be due to
utilization of different CD30 activating antibodies or differences in
the biologic properties of Karpas 299 cell lines, which could have been
altered during culture. We have published our findings on cutaneous
anaplastic large-cell lymphoma cell lines, which show either no
response or a proliferative response to CD30 activation.5
The cutaneous anaplastic large-cell lymphoma cell lines are also
responsive to NF-B and mitogen-activated protein kinase (MAPK)
inhibitors when used in combination with CD30-activating antibodies. We
conclude that NF-B is a key element in regulating CD30 responses and
that NF-B inhibitors could be used alone or in combination with CD30 agonistic antibodies in the treatment of Hodgkin disease and anaplastic large-cell lymphomas. But the matter is not as straightforward as
claimed in Mir et al1 since CD30 activation may also have cell-cycle-regulatory effects, as well as proapoptotic effects. Cell-cycle-inhibitory effects of CD30 activation require further investigation.
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| Figure 2.
Effects of CD30 activation on
NF-B activity.
(A) NF-B activity determined by the gel-shift assays. Karpas 299 cell line was incubated with HeFi-1 or isotype-specific control IgG for
1 hour. NF-B activity was determined by mobility shift observed in
the gel electrophoresis of nuclear extracts. The left lanes in
individual figures are loaded with radioactive labelled oligoprobes
representing NF-B binding sites and nuclear extracts (H). The right
lanes are loaded with radioactive probes plus excess amounts of
(× 200) unlabeled probes. These lanes represent the negative controls
for the assays and characterize which bands are specific for NF-B
binding activity (C). In the Karpas 299 cell line, there was no NF-B
activity after control IgG incubation; CD30 activation caused
activation of NF-B. Jurkat cell line nuclear extracts were used as
controls. N is the negative control, and P is the positive control. (B)
Effects of the NF-B inhibitor SN50 on systemic ALCL and Hodgkin
lymphoma cell lines. Proliferation assays were performed in the
presence of HeFi-1 and the NF-B inhibitor SN50 on the Karpas 299 and
KMH2 cell lines. SN50 alone caused inhibition of the KMH2 cell line,
whereas it had no effect on the Karpas 299 cell line. Combined
treatment with HeFi-1 and SN50 enhanced the inhibitory effects of
HeFi-1 on the Karpas 299 cell line but had no significant additional
effect on the KMH2 cell line. The results are presented as percentages
of 3H-thymidine incorporation values compared to controls
(IgG incubated). Each of 3 experiments was done in triplicate. A
representative experiment is shown.
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Edi Levi, Walther M. Pfeifer, and Marshall E. Kadin
Correspondence: Marshall E. Kadin, Department of Pathology, YA
309, Beth Israel-Deaconess Medical Center, 330 Brookline Ave, Boston,
MA, 02215; e-mail: mkadin{at}caregroup.harvard.edu
References
1.
Mir SS, Richter BWM, Duckett CS.
Differential effects of CD30 activation in anaplastic large cell lymphoma and Hodgkin disease cells.
Blood.
2000;96:4307-4312[Abstract/Free Full Text].
2.
Hecht TT, Longo DL, Cossman J, et al.
Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells.
J Immunol.
1985;134:4231-4236[Abstract].
3.
Pfeifer W, Levi E, Petrogiannis-Haliotis T, Lehmann L, Wang Z, Kadin ME.
A murine model for human CD30+ anaplastic large cell lymphoma: successful growth inhibition with an anti-CD30 antibody (HeFi-1).
Am J Pathol.
1999;155:1353-1359[Abstract/Free Full Text].
4.
Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J.
Inhibition of nuclear translocation of transcription factor NF-B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence.
J Biol Chem.
1995;270:14255-14258[Abstract/Free Full Text].
5.
Levi E, Wang Z, Petrogiannis-Haliotis T, et al.
Distinct effects of CD30 and Fas signaling in cutaneous anaplastic lymphomas: a possible mechanism for disease progression.
J Invest Dermatol.
2000;115:1034-1040[CrossRef][Medline]
[Order article via Infotrieve].
Response:
Strength of CD30 signal determines sensitivity to
apoptosis
Levi et al have reported that treatment of the anaplastic
large-cell lymphoma line Karpas 299 with the CD30-specific monoclonal antibody HeFi-1 does not induce apoptotic cell death.1,2 In contrast, under the conditions described in our paper,3 we readily observe apoptotic cell death in response to CD30 activation not only in the Karpas 299 line but also in a range of other
CD30-positive cell lines of anaplastic large-cell lymphoma origin. In
fact, our original decision to focus on the 2 CD30-sensitive cell lines Karpas 299 and Michel for the study described in our paper was based on
2 previous publications that demonstrated these lines' susceptibility
to cell killing by the CD30-agonistic antibodies M44 and
M67.4,5 Therefore, for our studies we used both M44 and
M67 and found very similar proapoptotic effects with these 2 antibodies. The experiments reported by Levi et al differ
substantially to those described in our paper. For example, the nuclear
factor (NF)-B status was evaluated by gel retardation one hour
following activation, whereas in our paper we used a reporter assay to
integrate B-directed reporter gene activity over a 36-hour period.
We feel it is important to note that our original model of CD30
regulation proposes that NF-B induction occurs almost immediately
after CD30 activation in a TRAF2-dependent fashion but that upon
prolonged stimulation TRAF2 is degraded and NF-B induction is
impaired.6 Therefore, the gel retardation experiments
performed by Levi et al and the reporter-gene analysis presented in our
paper are consistent with our model. We have extensively compared the experimental procedures in our paper
to those described in the above letter and in previous papers by Levi
and colleagues. Possibly the most significant difference is the
apparent use by Levi et al of antibody in its soluble form, whereas our
experiments were performed using immobilized antibody, as detailed in
"Materials and methods." To examine this issue, we have compared
the cytotoxic effects on Karpas 299 cells of the addition of antibodies
to CD30 in soluble or plate-bound form. Karpas 299 cells were added to
wells containing immobilized CD30-agonistic antibodies M67 and M44 or
an isotype control antibody, exactly as described in our original
paper. In parallel, these antibodies were provided in their soluble
form, as used previously by Levi and colleagues, to Karpas 299 cells at
2 different concentrations. Consistent with our earlier report,
incubation of Karpas 299 cells with immobilized antibodies to CD30 was
found to potently induce cell death, whereas in the same experiment the
addition of these antibodies in their soluble form did not induce
cell death and actually slightly enhanced viability (Figure
1). Also noteworthy is the fact
that, in the earlier report by Gruss et al4 in which the
cytotoxic effects of M44 and M67 were originally described, plate-bound
antibodies were used. In preliminary experiments using plate-bound
HeFi-1, we have also observed a proapoptotic effect (data not shown).
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| Figure 1.
Effects of plate-bound and soluble CD30-agonistic
antibodies on Karpas 299 cells.
Karpas 299 cells were treated for 20 hours either with plate-bound CD30
antibodies M44 and M67 (provided by Immunex Corporation), as indicated,
exactly as described previously,3 or with the soluble
antibodies at the indicated concentrations. Cell viabilities were
evaluated by propidium iodide exclusion and flow cytometry, and
normalized to the IgG1 isotype control as described
previously.3 The experiment was performed in triplicate,
and standard deviations are shown.
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In summary, we suggest that apparently different experimental
methods are the most likely explanation for the discrepancies between
our report and that of Levi et al, in particular the use of immobilized
antibody versus plate-bound antibody. The different effects of soluble
and immobilized antibody are suggestive of an intriguing physiologic
mechanism by which low levels of CD30 activation may induce cell-cycle
arrest, activation of NF-B, and the concomitant induction of
antiapoptotic genes, whereas a stronger CD30-activation signal may
result in a transient and more limited activation of NF-B and
ultimately cell death. The strength of the CD30 signal may be
determined by numerous factors, including the density of CD30 receptors
on the cell and the form of ligand (ie, membrane-bound or soluble). The
threshold sensitivity of a given cell may also be determined by
additional factors, such as the stability of intracellular signaling
intermediates, particularly TRAF2. Thus, the apparent discrepancies
between our data and those of Levi et al may reflect a novel
physiologic function of CD30.
Samy S. Mir, Bettina W. M. Richter, and Colin S. Duckett
Correspondence: Colin S. Duckett, Metabolism Branch, Division of
Clinical Sciences, National Cancer Institute, National Institutes of
Health, 10 Center Dr, Rm 6B-05, Bethesda, MD 20892-1578; e-mail:
duckettc{at}helix.nih.gov
References
1.
Levi E, Wang Z, Petrogiannis-Haliotis T, et al.
Distinct effects of CD30 and Fas signaling in cutaneous anaplastic lymphomas: a possible mechanism for disease progression.
J Invest Dermatol.
2000;115:1034-1040.
2.
Pfeifer W, Levi E, Petrogiannis-Haliotis T, Lehmann L, Wang Z, Kadin ME.
A murine xenograft model for human CD30+ anaplastic large cell lymphoma: successful growth inhibition with an anti-CD30 antibody (HeFi-1).
Am J Pathol.
1999;155:1353-1359.
3.
Mir SS, Richter BWM, Duckett CS.
Differential effects of CD30 activation in anaplastic large cell lymphoma and Hodgkin Disease cells.
Blood.
2000;15:4307-4312.
4.
Gruss H-J, Boiani N, Williams DE, Armitage RJ, Smith CA, Goodwin RG.
Pleiotropic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines.
Blood.
1994;83:2045-2056[Abstract/Free Full Text].
5.
Tian Z-G, Longo DL, Funakoshi S, et al.
In vivo antitumor effects of unconjugated CD30 monoclonal antibodies on human anaplastic large-cell lymphoma xenografts.
Cancer Res.
1995;55:5335-5441[Abstract/Free Full Text].
6.
Duckett CS, Thompson CB.
CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival.
Genes Dev.
1997;11:2810-2821[Abstract/Free Full Text].
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