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Experimental Therapeutics, Preclinical Pharmacology |
Division of Experimental Hematology, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-related apoptosis-inducing ligand (TRAIL). TRAIL
induced apoptosis within 4 h and also reduced clonogenic survival,
both reversible by caspase inhibitors. DR5 (but not DR4) was expressed
at high level in all cell lines. Expression of the decoy receptors DcR1
and DcR2 did not correlate with TRAIL sensitivity. All RMS lines
expressed the adapter molecule FADD, and six of seven expressed
procaspase-8. Expression of the inhibitory proteins c-FLIPL
and c-FLIPS was high in three TRAIL-sensitive (RD, Rh1, and
Rh30) and two TRAIL-resistant (Rh28 and Rh41) lines. All RMS lines
expressed Bid and procaspases-3, -6, -7, and -9.
Procaspases-8 and -10 were highest in TRAIL-sensitive RMS (RD, Rh1, and
Rh30), and procaspase-10 was not expressed in Rh18, Rh36, or Rh41.
TRAIL induced loss of mitochondrial membrane potential in
TRAIL-sensitive Rh1 but not in TRAIL-resistant Rh41 cells. There was no
correlation between expression of members of the Bcl-2 family (Bcl-2,
Bcl-xL, Bax, and Bak) and TRAIL sensitivity. TRAILsensitive Rh18
expressed procaspase-8 in the absence of procaspase-10 and c-FLIP, and
procaspase-10 was not detected in TRAIL-resistant Rh41 in the presence
of procaspase-8 and c-FLIP. Data suggest that caspase-8 may be
sufficient to deliver the TRAIL-induced apoptotic signal in the absence
of both caspase-10 and c-FLIP (Rh18) but not in the presence of c-FLIP
(Rh41). In RD, Rh1, and Rh30, the presence of c-FLIP may require
amplification of the apoptotic signal via caspase-10. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The only primary cells susceptible to apoptosis induction by the cytotoxic ligand TRAIL are activated T lymphocytes, suggesting a role in limiting the immune response (15 , 16) . In contrast, several tumors of diverse origin are sensitive to TRAIL. The receptors DR4 and DR5 (expressing two alternatively spliced isoforms), similar to Fas, contain death domains and can transmit a cell death signal. In contrast, two additional receptors (decoys), DcR1 (lacking a transmembrane or cytoplasmic domain) and DcR2 (containing a truncated death domain), bind the ligand with comparable affinity to DR4 and DR5, cannot transmit a cell death signal, and may protect normal cells from the cytotoxic action of TRAIL (17 , 18) . In neoplastic disease, DR4 and DR5 are expressed in primary human brain tumors (19) , melanomas (20) including those resistant to FasL (21) , and colon carcinomas (22) . Further, TRAIL has induced apoptosis in malignant glioma cell lines (23) , melanoma cell lines (20) , and breast and colon carcinoma cell lines (22) . After trimerization of the receptor, the pathway by which TRAIL induces apoptosis remains to be clearly defined.
RMS is the most common soft-tissue sarcoma in children under the age of 15, and despite aggressive approaches incorporating surgery, dose-intensive combination chemotherapy, and radiation therapy, the outcome for patients with metastatic disease remains poor (24 , 25) . Histologically, the vast majority of tumors demonstrate ERMS or ARMS histology, the latter being considerably more refractory to therapy. Hence, coupled with the relative rarity of this tumor in neoplasia, model systems become essential in delineating novel targets that may be important in developing new therapeutic strategies for treatment of RMS. In this study, only two of seven human RMS cell lines were partially sensitive to Fas-mediated apoptosis at high concentrations of anti-Fas (200 ng/ml). In contrast, four of seven RMS lines were highly sensitive to the cytotoxic ligand TRAIL at low concentrations of the cytotoxic ligand (1 ng/ml). TRAIL-sensitive lines were of both embryonal and alveolar histology. Upon examination of potential determinants of Fas sensitivity, levels of Fas expression were low, and expression of c-FLIP, an inhibitory protein that prevents recruitment of procaspase-8 to the DISC (26) , was high in five lines. In contrast, all RMS cell lines expressed high levels of the TRAIL receptor DR5, and TRAIL sensitivity was best correlated with the expression of procaspases-8 and -10. Data suggest that caspase-8 may be sufficient to deliver the TRAIL-induced apoptotic signal in the absence of both caspase-10 and c-FLIP, whereas in the presence of c-FLIP, amplification of the apoptotic signal may be required via caspase-10. These models of pediatric RMS may be important in the exploration of determinants of the TRAIL signaling pathway and apoptosis and in elucidating the role of TRAIL in the development of new therapeutic approaches for the treatment of RMS.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growth Inhibition and Clonogenic Assays.
For growth inhibition assays, cells were plated at a density of 100,000
cells/well in six-well plates in 2 ml of medium. After overnight
attachment, cells were treated for 72 h or 7 days (Rh28) with the
cytolytic anti-Fas MoAb CH-11 (50–200 ng/ml; MBL International Corp.)
or the cytotoxic ligand TRAIL (Alexis Biochemicals; 0.5–100 ng/ml, in
the presence of 0.25 µg/ml of an anti-FLAG enhancer MoAb). At the
beginning of and after treatment, cells were enumerated using a Coulter
particle counter. Inhibition of cell growth compared with control
growth was determined, and the percentage of cells remaining at the end
of treatment was also evaluated (% time 0).
For clonogenic assays, Rh1 and RD cell lines were plated at a density of 2000 or 3000 cells/well, respectively, in 2 ml of medium. After overnight attachment, cells were treated with TRAIL (1–100 ng/ml) for 72 h either in the absence or presence of a chimeric fusion protein, DR5-Fc (50 ng/ml; Alexis Biochemicals), that prevents the binding of TRAIL to its receptors. Clonogenic survival was determined after a further 7 days, as described previously (4) . Additionally, cells were treated for 16 h with TRAIL (25 ng/ml) either in the absence or in the presence of the caspase inhibitors (10 µM, 20 µM; Enzyme Systems Products). These included Z-VAD-FMK (general inhibitor), Z-DEVD-FMK (inhibitor of caspases 3,6,7,8,10), Z-IETD-FMK (caspase-8 inhibitor), or a negative control (Z-FA-FMK), with subsequent determination of clonogenic survival as described.
Measurement of Apoptosis.
Rh1 and RD cells were plated at a density of 400,000 cells/well in
six-well plates. After overnight attachment, cells were treated for
periods up to 24 h with CH-11 or TRAIL either in the absence or
presence of caspase inhibitors. Both the floating cells and attached
cells were pooled after trypsinization, fixed in 70% ethanol, and
stored at -20° prior to analysis. Apoptotic cells were detected as a
sub-G1 fraction after propidium iodide staining
and analysis using a Becton Dickinson FACScan (30
, 31) .
RNase Protection Analysis.
Total RNA was extracted from 20 x 106 cells
in RNAzol B (Tel-test) using standard procedures. The RiboQuant
Multi-Probe RNase protection assay was performed according to the
vendor (PharMingen). Three multiprobe template panels were used: hAPO-3
(procaspase-8, Fas, FADD, FasL, FAF, FAP, TNR60, TRADD, TRAIL, DR3, and
RIP); hAPO-3c (procaspase-8, Fas, FasL, DcR1, DR3, DR4, DR5, TRAIL
TNFR60, and TRADD); and hAPO-1c (procaspases-1 to -10).
RT-PCR.
The expression of c-FLIP (c-FLIPL and
cFLIPS), procaspase-8, procaspase-10, and the
TRAIL decoy receptor DcR2 was also determined by semiquantitative
RT-PCR, as described previously (3)
. Complimentary DNA was
synthesized from 2 µg of total RNA in a 20-µl reaction using an
oligo(dT) primer and a cDNA cycle kit (Invitrogen). ß-actin was used
as a control to monitor RT-PCR amplification efficiency and quality of
the cDNA from 2 µl of the template at 25 cycles, as reported
previously (3)
. PCR products were separated by
electrophoresis in a 2% agarose gel and visualized by ethidium bromide
staining and UV light illumination. The primers used for PCR of
procaspase-8 (273 bp) were as described (3)
and for
procaspase-10 (500 bp), c-FLIPL (501 bp),
c-FLIPS (302 bp), and DcR2 (369 bp) were as
follows: CASP-10F, 5'-CATAGGATTGGTCCCCAACA-3'; CASP-10R,
5'-CTTGATACGACTCGGCTTCC-3'; c-FLIPLF,
5'-CTTGGCCAATTTGCCTGTAT-3'; c-FLIPLR,
5'-GGCAGAAACTCTGCTGTTCC-3'; c-FLIPSF,
5'-CGAGGCAAGATAAGCAAGGA-3'; cFLIPSR,
5'-CACATGGAACAATTTCCAAGAA-3'; DcR2F, 5'-CGGAAGAAATTCATTTCTTACCTC -3';
and DcR2R,5'-TTCTTCCAGTGTTGCCGAGGC-3'.
Western Analysis.
Gene expression was also examined by Western analysis
(14)
. Primary antibodies used were: procaspase-8 (MBL),
procaspase-3 (PharMingen), FADD (Transduction Laboratories), Bid (Santa
Cruz Biotechnology), and NF6 for the detection of
c-FLIPL, and c-FLIPS was a
generous gift from Dr. Peter Krammer (German Cancer Research Center,
Heidelberg, Germany). Secondary antibodies were horseradish
peroxidase-conjugated goat antimouse IgG1 and rabbit antigoat IgG from
Southern Biotechnology and sheep-antimouse immunoglobulin and donkey
antirabbit immunoglobulin from Amersham. Signal was detected using the
ECL Western blotting system (14)
.
Flow Cytometric Analysis.
The specific binding of TRAIL to cell surface receptors was determined
in TRAIL-sensitive RD and TRAIL-resistant Rh36 cell lines by measuring
the binding of TRAIL in the presence of an anti-FLAG enhancer mAb and
secondary antimouse-IgG1 FITC Ab using flow cytometric analysis as
described previously (20)
.
Measurement of Mitochondrial Membrane Potential (
).
Rh1 and Rh41 cell lines were plated at a density of 2 x
105 cells/well in six-well plates. After
overnight attachment, cells were treated, in duplicate, with 50 ng/ml
TRAIL (Alexis) and 500 ng/ml enhancer (Alexis) for 16 h. At the
end of incubation, both the floating cells and attached cells were
pooled after trypsinization. Subsequently, the cells were incubated
with the dye JC-1 (Alexis Biochemicals; 10 µg/ml) in 1 ml of medium
for 10 min at 37oC, washed in PBS, and analyzed
by flow cytometry (FACS-Scan; Becton Dickinson); 10,000 cells were
subsequently collected and analyzed by CellQuest Software as described
previously (32, 33, 34)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cells were
exposed to anti-Fas at concentrations from 50–200 ng/ml, and data
derived at the highest concentration are shown. All cell lines were
relatively resistant to induction of apoptosis via Fas. Anti-Fas
induced partial growth inhibition in Rh1 and Rh18 after 72 h
exposure. This was related to the induction of apoptosis in a fraction
of the two cell populations (30%) within 24 h (data not shown).
However, the overall effect was growth inhibitory only and did not
result in loss of cell numbers at the end of anti-Fas exposure because
the surviving cells repopulated (Fig. 1)
, in contrast to 100% cell
kill determined in Fas-sensitive GC3/c1 human
colon carcinoma cells (3)
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. Growth inhibition was determined in
RD, Rh1, Rh18, and Rh30, whereas Rh28, Rh36, and Rh41 were resistant to
TRAIL. In the four TRAIL-sensitive cell lines, all demonstrated fewer
surviving cells at the end of 72 h in comparison with the cell
numbers at the start of treatment, indicating that TRAIL was cytotoxic.
The exquisite sensitivity of the four cell lines to TRAIL was evident
from effects demonstrated at concentrations of 
1 ng/ml. In
TRAIL-sensitive RD and Rh1 cell lines, the effect of 72 h exposure
to varied concentrations of TRAIL on clonogenic survival was determined
either in the absence or presence of the chimeric fusion protein DR5-Fc
(50 ng/ml; Fig. 3
). Loss in clonogenic
survival of 90% was demonstrated at 6 ng/ml TRAIL, and significant
protection from TRAIL-induced cytotoxicity was demonstrated after
coincubation with DR5-Fc.
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. Rapid
induction of apoptosis was observed. By 4 h, almost 40% of the
cells were undergoing apoptosis, and by 12 h, a plateau was
reached where 70% of the cells were apoptotic. The effect of caspase
inhibitors on the induction of apoptosis was subsequently examined
after 16 h exposure to TRAIL (25 ng/ml; Fig. 5
). In TRAIL-treated Rh1 cells, 
80%
of the cells were apoptotic at this time. However, in the presence
of either Z-VAD-FMK, Z-DEVD-FMK, or Z-IETD-FMK, apoptosis was
completely inhibited. Furthermore, coincubation of Rh1 cells with each
of the three caspase inhibitors also reduced the loss in clonogenic
survival after 16 h of TRAIL exposure (Fig. 6)
.
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and/or by RT-PCR (Fig. 8)
.
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Of importance to TRAIL-induced apoptosis in RMS cell lines is that all
lines demonstrated high levels of expression of DR5 by RNase protection
analysis (Fig. 7)
. In contrast only Rh1 expressed high levels of DR4.
Of the two decoy receptors, DcR1 was detected at low levels in Rh1
(TRAIL-sensitive) and Rh28 (TRAIL-resistant; Fig. 7
), whereas DcR2 was
detected in TRAIL-sensitive lines Rh1 and Rh30 and in TRAIL-resistant
Rh28 (Fig. 8)
. Hence, the expression of either decoy receptor failed to
correlate with the cellular resistance of RMS cell lines to TRAIL.
Surface expression of TRAIL receptors was examined by flow cytometric
analysis by measuring the binding of TRAIL, in TRAIL-sensitive RD and
TRAIL-resistant Rh36 cell lines, that expressed DR5 only and no decoy
receptors (Fig. 9)
. Binding of TRAIL to
the cell surface was determined to be similar in each of the two cell
lines and correlated with expression of DR5 mRNA.
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.
Although TRAIL-resistant Rh28 and Rh41 expressed c-FLIP, similar levels
of expression of both isoforms were found in TRAIL-sensitive RD, Rh1,
and Rh30. Of the caspases that may be important in the regulation of
TRAIL-induced apoptosis, including caspases-3, -6, -7, and -9, these
were detected in all of the seven RMS cell lines. However, the
initiator procaspases-8 and -10 demonstrated the greatest differential
in expression among the seven cell lines (Fig. 7)
and were further
examined by RT-PCR (Fig. 8)
. Procaspase-8 was present in all lines
except for Rh36 (TRAIL-resistant), was low in expression in
TRAIL-resistant Rh28, and highest in expression in RD, Rh1, and Rh30,
which demonstrated the greatest sensitivity to TRAIL. Procaspase-10 was
expressed in TRAIL-sensitive RD, Rh1, and Rh30 and in TRAIL-resistant
Rh28, using primers that detect all procaspase-10 isoforms as a single
PCR product (Fig. 8)
. Of interest, procaspase-10 was not expressed in
TRAIL-sensitive Rh18, although this line expressed procaspase-8 in the
absence of c-FLIP. Furthermore, procaspase-10 was not expressed in
TRAIL-resistant Rh41 in the presence of procaspase-8 and c-FLIP, and
TRAIL-resistant Rh36 failed to express either procaspases-8 or -10.
The expression of procaspase-8, procaspase-3, FADD,
c-FLIPL, and c-FLIPS was
also examined by Western analysis. Levels of expression of proteins
correlated with levels of expression of the respective mRNAs (Fig. 10)
. Furthermore, Bid (Fig. 11)
, which is cleaved by caspase-8
prior to mitochondrial membrane insertion, was expressed in all cell
lines.
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). In Rh1, >80% of cells
demonstrated low membrane potential after TRAIL treatment, whereas
there was no significant change detected in Rh41. To investigate
whether the threshold for mitochondrial membrane depolarization may
determine sensitivity to TRAIL, the expression of members of the Bcl-2
family with both antiapoptotic (Bcl-2 and Bcl-xL) and proapoptotic (Bax
and Bak) function that function at the level of the mitochondria was
examined (Fig. 13)
. The expression of
Bcl-2 and Bak was almost identical in the seven RMS lines. Bcl-xL
expression was high in cell lines sensitive to TRAIL, and Bax was
expressed in high levels in TRAIL-sensitive lines RD, Rh1, Rh18, and
Rh30 but also in TRAIL-resistant Rh41. Thus, there was no direct
correlation between changes in mitochondrial membrane potential and
sensitivity to TRAIL.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The anti-apoptotic factor, c-FLIP, exists in two isoforms, c-FLIPS and c-FLIPL, which appears to be the predominant form expressed in mammalian cells (26 , 35) . After Fas triggering, FADD, procaspase-8, and c-FLIP isoforms are recruited to the Fas receptor complex. c-FLIP subsequently inactivates the DISC by blocking further recruitment of procaspase-8 into the complex, thereby inhibiting the activation of caspase-8 (14 , 26 , 35) . In cells with high Fas expression, the presence of c-FLIP appears insufficient to block apoptosis. However, in cells with low receptor expression, this is a factor in cellular resistance to the induction of apoptosis via Fas (14) . The level of Fas expressed in Rh1 is lower than reported in TS- and Thy4 human colon carcinoma cells, highly sensitive to Fas-induced apoptosis (3) . Therefore, c-FLIP may be a factor in the Fas-resistance phenotype in Rh1, because procaspase-8 and FADD were both expressed. The second RMS cell line to demonstrate a partial growth-inhibitory response to anti-Fas was Rh18, and although this cell line expressed low levels of Fas, this was in the absence of c-FLIP expression.
In contrast to the general lack of sensitivity of RMS cell lines to
Fas-induced apoptosis, four RMS cell lines were highly sensitive to the
cytotoxic ligand TRAIL. All lines demonstrated high-level expression of
one of the receptors, DR5, that expresses a death effector domain
thereby allowing transmission of an apoptotic signal. Four RMS cell
lines (RD, Rh1, Rh18, and Rh30) were sensitive to TRAIL at
concentrations 1 ng/ml, which also reduced clonogenic survival, and
three lines were TRAIL resistant (Rh28, Rh36, and Rh41). Expression of
the decoy receptors, DcR1 and DcR2, demonstrated no correlation with
the sensitivity of RMS cell lines to TRAIL. Thus, the lack of
sensitivity to TRAIL-induced apoptosis in Rh28, Rh36, and Rh41
suggested that factors other than expression of receptors determined
the resistance phenotype.
c-FLIP can inhibit TRAIL-induced apoptosis (20 , 36) in addition to Fas-mediated apoptosis. To date, limited information is available concerning the receptor complexes for the TRAIL receptors DR4 and DR5. Certain studies have reported direct binding of the adapter proteins FADD and TRADD to these receptors and inhibition of TRAIL-induced apoptosis in the presence of DN-FADD and DN-TRADD (20) . Furthermore, DISC formation among DR4 or DR5, FADD, and caspase-8 has recently been identified (37, 38, 39) , which could explain an inhibitory role for c-FLIP in TRAIL-induced apoptosis. However, the exact mechanism of receptor complex formation for DR4 and DR5 including potential adapters other than FADD, and activation of specific caspases in the mechanism of TRAIL-induced apoptosis have yet to be critically identified. All RMS cell lines expressed FADD, and six of seven expressed procaspase-8. Furthermore, expression of c-FLIP did not correlate directly with lack of sensitivity to TRAIL in RMS cell lines, because TRAIL-resistant Rh36 did not express c-FLIP, and TRAIL-sensitive RD, Rh1, and Rh30 cell lines expressed both isoforms. These results are in contrast to those reported by Kim et al. (40) , who suggested that DR4 and c-FLIP expression could be potentially useful predictors of TRAIL sensitivity. Of specific interest in RMS cell lines was the observation that in RD, Rh1, and Rh30, procaspase-10 was expressed in addition to procaspase-8 in the presence of high levels of c-FLIP.
In Fas-mediated apoptosis, two signaling pathways have been demonstrated (14 , 41) . In type I proposed by Scaffidi et al. (14) , ligation of Fas leads to strong caspase-8 activation at the DISC, thereby directly activating other caspases including caspase-3 in the absence of mitochondrial involvement. In type II Fas-mediated cell death, only a small amount of the DISC is formed, leading to the activation of mitochondria, which amplifies the signal when caspase-8 cleaves the cytosolic substrate Bid, leading to release of mitochondrial cytochrome c and activation of caspases-3, -7, -8, and -9 (14 , 41) , a process that can be blocked by antiapoptotic members of the Bcl-2 family (41) . RMS cell lines in this study expressed procaspases-3, -6, -7, -8, and -9.
The procaspases activated by TRAIL still remain to be defined, although caspase-8 is activated during formation of a TRAIL-induced DISC (37, 38, 39) The recent ordering of the caspase signaling cascade downstream of the mitochondria demonstrates activation of caspase-9, followed by caspase-3 and caspase-6, which further activates caspases-8 and -10 (42) . It is evident that TRAIL induced mitochondrial membrane depolarization in TRAIL-sensitive Rh1 but not in TRAIL-resistant Rh41 RMS cell lines. However, the relative expression levels of both proapoptotic (Bax and Bak) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family that function at the level of the mitochondria did not correlate with the effect of TRAIL on mitochondrial membrane potential. Furthermore, Bid expression was similar among the seven RMS cell lines, suggesting that it may be factors upstream of the mitochondria that may determine the sensitivity of RMS cell lines to TRAIL. Activation of both caspase-8 and caspase-3 have been identified downstream of the receptors in TRAIL-induced apoptosis in human melanoma cell lines (20) , although activation of caspase-10 was not examined. Of interest was that expression of procaspases-8 and -10 were highest in TRAIL-sensitive RMS cell lines. Little information is available concerning the function of caspase-10 (43) . However, it is known that the functional death effector domain binds to the corresponding domain of FADD (44) , suggesting that FADD may be the adapter protein for procaspase-10. Furthermore, procaspase-10 is expressed in high level in fetal skeletal muscle cells but is low or absent in this tissue in the adult, suggesting a role for caspase-10 in development (43) . Hence, procaspase-10 expressed in malignant RMS cells that resemble fetal muscle myoblasts could be activated in response to stress. Of interest in RMS cell lines was that procaspase-8, but not procaspase-10 or c-FLIP, was expressed in TRAIL-sensitive Rh18, and in TRAIL-sensitive RD, Rh1, and Rh30 c-FLIP isoforms were expressed at high level in addition to procaspase-8 and procaspase-10. Furthermore, procaspase-10 was not expressed in two of three TRAIL-resistant lines. It is possible in TRAIL-sensitive lines with high c-FLIP expression that caspase-10 activation is necessary for the induction of apoptosis downstream of caspase-8 activation for amplification of the apoptotic signal, whereas in Rh18, caspase-8 activation in the absence of c-FLIP expression is sufficient to induce apoptosis directly. Caspase activation was clearly important in the mechanism of TRAIL-induced apoptosis in RMS cell lines because all caspase inhibitors blocked not only TRAIL-induced apoptosis but also TRAIL-induced loss in clonogenic survival. The role of caspase-8 in TRAIL-induced apoptosis in RMS may be further substantiated by the effectiveness of the caspase-8 inhibitor Z-IETD-FMK in this regard. However, the relative roles of caspase-8 and caspase-10 may be further defined in these models of pediatric RMS because of the differences in their cellular expression and TRAIL sensitivity among the different RMS cell lines.
It has been well documented that RMS with alveolar histology are less sensitive to therapeutic modalities than ERMS. Furthermore, high mortality rates remain in patients with metastatic disease (24 , 25) . There is, therefore, a need to continue to identify new targets and new therapeutic approaches for the treatment of patients with RMS. Of significance is that both ERMS and ARMS cell lines demonstrated sensitivity to the cytotoxic ligand TRAIL, as well as high DR5 expression, irrespective of the pathological diagnosis. On the basis of the similar sensitivity of ERMS and ARMS cell lines to TRAIL, it will be of importance to determine whether the approach of using TRAIL as a cytotoxic agent may be further exploited in the therapy of RMS.
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FOOTNOTES |
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1 Supported by NIH Awards PO1 CA 23099, the Cancer
Center Support (CORE) Grant CA 21765, and by the American Lebanese
Syrian Associated Charities. I. P. was supported in part by a
scholarship from the Fulbright Program and by the First Institute of
Pathology and Experimental Cancer Research, Semmelweiss University of
Medicine, Budapest, Hungary. 
2 To whom requests for reprints should be
addressed, at Division of Experimental Hematology, Department of
Hematology-Oncology, St. Jude Children’s Research Hospital, 332 North
Lauderdale, Memphis, TN 38105. Phone: 901-495-3456; Fax: 901-523-2622;
E-mail: janet.houghton{at}stjude.org
3 The abbreviations used are: FADD, Fas-associated
death domain; DISC, death-inducing signaling complex; TRAIL, tumor
necrosis factor--related apoptosis-inducing ligand; FasL, Fas
ligand; RMS, rhabdomyosarcoma; ERMS, embryonal RMS; ARMS, alveolar RMS;
MoAb, monoclonal antibody; RT-PCR, reverse transcription-PCR; FACS,
fluorescence-activated cell sorter.
Received 3/15/00; revised 8/ 2/00; accepted 8/ 2/00.
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