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Regular Articles |
From the Departments of Pathology*
and Bacterial
Infection,§
Institute of Medical Science,
University of Tokyo, Tokyo, Japan; the First Department of Internal
Medicine
and the Department of
Pathology,¶
Faculty of Medicine, University of
Tokyo, Tokyo, Japan; the Developmental Oncology/Hematology and Leukemia
Unit,
Department of Medical Oncology, Centro
di Riferimento Oncologico, I.R.C.C.S., Aviano, Italy; the Clinical
Laboratory,||
National Children’s Hospital, Tokyo, Japan;
and the Fourth Department of Internal Medicine,**
Kitasato University, School of Medicine, Kanagawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
(NF-B) as CD30, and its overexpression in HL-60 induced a
differentiated phenotype. To better understand the physiological and
pathological functions of CD30v, expression of this variant was
examined using a multiple approach to examine 238 samples of human
malignant myeloid and lymphoid neoplasms. Screening by reverse
transcriptase-polymerase chain reaction (RT-PCR) revealed expression of
CD30v transcripts in 52 of 72, 7 of 11, 63 of
90, and 7 of 30 samples of acute myeloid leukemia
(AML), myeloid blast crisis of myeloproliferative disorders
(MBC), and lymphoproliferative disorders (LPDs) of B- and
T-cell origin, respectively. CD30v expression was high in
monocyte-oriented AMLs (FAB M4 and M5), B-cell chronic
lymphocytic leukemia (B-CLL), and multiple myeloma (MM). Using
the specific antibody HCD30C2, prepared using a peptide
corresponding to the nine amino acids of the amino-terminal
CD30v, expression of CD30v protein was detected in 10 of 25 and
2 of 10 AML and ALL samples, respectively. In AMLs,
immunocytochemical detection of CD30v revealed the presence of loose
clusters of CD30v-expressing cells dispersed amid a population of
CD30v-negative blasts. Finally, the parallel expression of
CD30v mRNA and protein, as evidenced by Northern and Western
blotting, was confirmed in selected cases of AMLs and LPDs. A
significant correlation was found between expressions of CD30v and CD30
ligand transcripts in AML and LPD (P =
0.02, odds ratio = 3.2). The association of CD30v with
signal-transducing proteins, tumor necrosis factor
receptor-associated factor (TRAF) 2, and TRAF5 was demonstrated
by coimmunoprecipitation analysis, as was demonstrated for
authentic CD30 protein. Expression of transcripts for TRAF1,
TRAF2, TRAF3, and TRAF5, as demonstrated by
RT-PCR, was noted in leukemic blasts that express CD30v.
Collectively, frequent expression of CD30v along with TRAF
proteins in human neoplastic cells of myeloid and lymphoid origin
provide supportive evidence for biological and possible pathological
functions of this protein in the growth and differentiation of a
variety of myeloid and lymphoid cells.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, LT-ß, CD27L, 4–1BBL, OX40L, and CD95L/FasL,
has effects on CD30-expressing cells, including activation,
proliferation, differentiation, and cell death, depending on cell type,
stage of differentiation, transformation status, and the presence of
other stimuli.5-7
Expression of CD30 ligand (CD30L) was noted in activated T cells,
resting neutrophils, eosinophils, and B cells, as well as in the cells
of various human malignant myeloid and lymphoid
neoplasms.5,8-10
Cross-linking of cell-surface CD30L by
an antibody or a soluble CD30 (CD30-Fc) can transduce signals leading
to gene expression and metabolic activation in granulocytes and T
cells.11
On the other hand, cross-linking CD30 induced
Ca2+ influx in T-cell lines,12
and signals
mediated by CD30 were seen to regulate gene expression through
activation of the nuclear factor-B (NF-B).13,14
We
and other investigators have demonstrated that CD30 binds to tumor
necrosis factor receptor-associated factor (TRAF) proteins TRAF1, 2,
and 5 in the C-terminal region.15-20
We recently reported
that the C-terminal cytoplasmic region has three independent NF-B
activating subdomains, all of which can function independently. The
C-terminally located two subdomains serve as TRAF binding domains, but
the most N-terminal subdomain can activate NF-B, without interacting
with TRAF proteins.21
A variant form of CD30 (CD30v), which retains only the cytoplasmic
domain and can mediate signals to activate NF-B, was identified in
our laboratory at the University of Tokyo.22
CD30v
expression was induced by phorbol ester in a human myeloid leukemia
cell line HL-60 and is constitutively expressed in alveolar
macrophages. Overexpression of the CD30v activated NF-B and induced
NBT reduction activity in HL-60 cells, findings that suggested a role
for this molecule in the activation and/or differentiation of myeloid
cells.
In the present study, we investigated expression of CD30v mRNA and protein in a broad series of primary human neoplastic cells of myeloid and lymphoid origin, using a combination of reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blotting, and immunostaining with a specific anti-CD30v polyclonal antibody raised against the amino-terminal peptide of CD30v. CD30v was frequently expressed in malignant cells of myeloid and B-lymphoid phenotypes, whereas a more restricted expression of CD30v was found in T-cell tumors. This distribution significantly correlated with expression of the physiological CD30-specific ligand CD30L. The in vivo interaction of CD30v with TRAF2 and TRAF5, as well as expression of transcripts for TRAF proteins in CD30v-expressing blast cells, was also evident. CD30v may possibly have a role in growth regulation in the case of human malignant myeloid and lymphoid neoplasms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our study included cells from peripheral blood (PB) or bone marrow (BM) of 203 patients with acute myeloid leukemias (AMLs) (n = 72), myeloid blast crisis (MBC) of chronic myeloproliferative disorders (n = 11), B- and T-cell lymphoproliferative diseases (n = 120), including B- and T-lineage acute lymphoblastic leukemias (ALLs), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HCL), high- and low-grade non-Hodgkin’s lymphomas (NHLs), multiple myeloma (MM), and adult T-cell leukemia (ATL) caused by HTLV-1. Diagnoses were based on cell morphology, immunophenotyping, enzyme cytochemistry, and clinical parameters, according to guidelines of international consensus.10,23-25 The cells were either immediately used for RNA extraction or frozen in RPMI 1640 containing 10% fetal calf serum (FCS) and 10% dimethyl sulfoxide and stored at -80°C until use. In addition, 35 formalin-fixed, paraffin-embedded BM samples from AML patients or ALL patients were also studied.
Cell Lines
The HL-60, 293T, and COS-7 cell lines were obtained through the Japanese Cancer Research Resources Bank (Tokyo, Japan) and Fujisaki Cell Biology Center (Okayama, Japan), and the Hodgkin’s disease (HD)-derived cell lines L428 and KM-H2, as well as the HEL, HUT-102, Karpas 299, and K-562 cell lines, were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). With the exception of 293T and COS-7 cell lines, cultured in Dulbecco’s minimum essential medium supplemented with 10% FCS, all other cell lines studied were maintained in RPMI 1640 containing 10% FCS. When indicated, HL-60 cells were treated with 50 ng/ml of 12-O-tetradecanoylphorbol 13-acetate (TPA), as reported.22
Cell Isolation and Purification
Anticoagulated PB and BM aspirates were collected from leukemia/lymphoma patients before any therapy and after informed consent was obtained. Neoplastic cells were isolated by centrifugation on a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient and, with the exclusion of T-cell-derived malignancies, were further purified by T-cell depletion with anti-CD2 immunomagnetic beads (Dynabeads; Dynal, Oslo, Norway).10 In the case of MM, tumor cells were further purified by positive indirect immunoselection with the plasma cell-specific monoclonal antibody BB-4.26 After purification procedures, all of the samples contained over 95% neoplastic cells.
RNA Isolation and RT-PCR
Total RNA was isolated from cell lines and leukemic cell samples with Isogen (Nippon-Gene, Toyama, Japan) or by the guanidinium thiocyanate method.27 Template cDNA was synthesized from 1 µg of total RNA with a Ready-To-Go T-Primed First-Strand kit (Pharmacia) or by avian myeloblastosis virus (AMV) reverse transcriptase (Promega Co., Madison, WI) in a 20-µl reaction mix. For each PCR, 2.0 or 2.5 µl was used as the template cDNA. All cDNAs were checked for first-strand synthesis with a primer pair specific for ß-actin. The primer pairs for CD30v are specific for CD30v, being the sense primer located within the unique nucleotide sequence of CD30v cDNA.22 Sequences and nucleotide positions of primers are as follows: CD30v: (sense) 5'-GAGTGTGGGGCGTCTCTGTG-3' (nucleotide position 27–46) or 5'-AGTGTGGGGCGTCTCTGTGTTC-3' (nucleotide position 28–49), (antisense) 5'-GTTCTGTCTCCTGCTCGGGGTAGT-3' (nucleotide position 598–575) or 5'-CGCTCCTCAGCTGCGTTGAG-3' (nucleotide position 228–247); TRAF1: (sense) 5'-GCCCCTGATGAGAATGAGTT-3' (nucleotide position 109–128), (antisense) 5'-CTCATGCTCTTGCACAGACT-3' (nucleotide position 380–399)28 ; TRAF2: (sense) 5'-ACAAGTGTGAGAAGTGCCAC-3' (nucleotide position 302–321), (antisense) 5'-GACGCACACATGGAAGTTCT-3' (nucleotide position 582–601)28 ; TRAF3: (sense) 5'-TTTGTCCCTGAACAAGGAGG-3' (nucleotide position 311–330), (antisense) 5'-AACTGCTCTGCACAACCTCT-3' (nucleotide position 581–600)29 ; TRAF5: (sense) 5'-GACTTTGAGCCCAGTATAGA-3' (nucleotide position 130–149) (antisense) 5'-CCCAGAATAACCTTCCCATT-3' (nucleotide position 403–422)30 ; ß-actin: (sense) 5'-ATGGATGATGATATCGCCGCG-3' (nucleotide position 42–62), (antisense) 5'-TTCTCCATGTCGTCCCAGTTG-3' (nucleotide position 272–292).31 Primer pairs specific for CD30 and CD30L were as reported elsewhere.10 Hot-start RT-PCR reactions were performed with Hot Wax beads (Invitrogen, Carlsberg, CA) after the condition for each primer set was optimized. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. In the case of CD30v, 10 µl of amplified cDNAs was run on 1.5% agarose gels, blotted onto nylon membranes (Boehringer Mannheim, Mannheim, Germany), and hybridized with 2 x 106 cpm/ml of 5'-end-labeled oligomer probes. Sequences and nucleotide positions of probes are as follows: CD30v probe 1 (recognizing CD30vL alone): 5'-GTCCAGACCTCCCAGCCCAAG C-3' (position 175–196 of CD30v); CD30v probe 2 (recognizing both CD30vL and S forms): 53-CCTTCCTGAGCCCCGGGTGTCCA-3' (position 396–418 of CD30v).
To exclude the possibility that CD30v primer pairs amplify from genomic DNA a fragment of the same size as that from cDNA templates, PCR was carried out on genomic DNA templates as well as on non-reverse-transcribed RNA and cDNAs synthesized from TPA-treated HL-60 RNA after treatment with RNase-free DNaseI (Promega).
Northern Blot Analysis
Northern blot analysis was carried out essentially as described.10 Briefly, 10 µg/lane of total RNA, extracted using the guanidinium thiocyanate method, were size-fractionated on 1% agarose gels containing 6.7% formaldehyde and subsequently blotted onto nylon membranes (Boehringer Mannheim). Filters were hybridized in 1.0 mol/L NaCl and 1% sodium dodecyl sulfate (SDS) at 68°C with 1.0 x 106 cpm/ml of random primed-labeled probes.10 After washings to a final stringency of 0.2x standard sodium citrate and 0.1% SDS at 65°C, filters were exposed to XAR-5 films (Eastman Kodak, Rochester, NY) at -80°C. The probes used were as follows: a 980-bp CD30-specific cDNA fragment obtained by cloning in pGEM-T vector (Promega), a RT-PCR product from the HD-derived cell line KM-H2, and an 838-bp human ß-actin-specific cDNA fragment obtained by cloning in the same vector a RT-PCR product from hemopoietic cell lines.
Preparation of Antiserum
Anti-CD30v polyclonal antibodies were generated essentially as reported.32 However, at variance with a previously reported antiserum obtained by immunizing rabbits with the whole CD30v molecule expressed as fusion protein,22 the present antiserum was generated by immunizing rabbits with an oligopeptide (MSQPLMETC) corresponding to the nine N-terminal amino acids of CD30v. Immune sera were affinity-purified over antigen columns prepared with CH Sepharose (Pharmacia); the purified antibody was named HCD30C2.
Immunoprecipitation and Immunoblotting
Immunoprecipitation and immunoblotting experiments using anti-CD30v or anti-CD30 (Ber-H2; DAKO Japan, Kyoto, Japan) antibodies were done as described.22 Briefly, COS-7 cells transfected with expression vectors for CD30v or CD30 were lysed in SDS-RIPA buffer (0.1% w/v SDS, 10 mmol/L Tris-HCl, pH 7.4, 1% v/v Nonidet P-40 (NP40), 0.1% w/v sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml aprotinin) on ice for 10 minutes and centrifuged at 15,000 rpm at 4°C for 20 minutes, and protein concentrations of the supernatants were measured using a Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA). Samples of cell lysates (100 µg) were incubated with HCD30C2 or Ber-H2 at 4°C for 60 minutes, and immune complexes were absorbed to protein G-Sepharose (Pharmacia), washed thoroughly with RIPA buffer, boiled with Laemmli’s sample buffer, and separated using an SDS-polyacrylamide gel (PAGE). Then the samples were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), followed by blocking for 12 hours with 10% w/v skim milk in Tris-buffered saline (TBS) at 4°C, and then incubated with 5 µg/ml HCD30C2 or Ber-H2 for 1 hour at 4°C. Peroxidase-conjugated sheep anti-rabbit or anti-mouse immunoglobulin antibodies (Amersham, Buckinghamshire, England) were used to detect immunoreactive proteins, using an ECL kit (Amersham). In some cases, cell lysates (100 µg) of primary leukemic samples and HL-60 cells treated with TPA or not, were directly subjected to 12% SDS-PAGE, transferred onto PVDF membranes, and sequentially incubated with the HCD30C2 antiserum and a peroxidase-conjugated secondary antibody, as described above.
Indirect Immunofluorescence Cytochemistry and Immunohistochemistry
Indirect immunofluorescence study was done using COS-7 cells transfected with expression vectors for CD30v or CD30, untreated HL-60 cells, and those treated with 50 ng/ml TPA and primary bone marrow-derived leukemic cells. Briefly, cells were grown on Lab-Tek Chamber Slides (Nunc, Naperville, IL) or plated on slide glass coated with poly-L-lysine hydrobromide (Sigma Chemicals, St. Louis, MO) by Cytospin 2 (Shandon, Pittsburgh, PA) and fixed for 10 minutes in 4% paraformaldehyde. After blocking with 10% v/v normal swine serum in phosphate-buffered saline (PBS) for 15 minutes, cells were incubated with the primary antibody (HCD30C2 or Ber-H2) for 1 hour at room temperature. After extensive washings in PBS, cells were incubated with fluorescent isothiocyanate (FITC)-conjugated isotype-matched immunoglobulins (TAGO, Burlingame, CA) for 30 minutes at room temperature and examined under a Nikon fluorescence microscope (Nikon, Tokyo, Japan).
Immunocytochemistry of CD30v was performed using an avidin-biotin-complex technique on BM samples from leukemia patients and normal controls and the HCD30C2 antibody. Sections of 4 µm were prepared from formalin-fixed and paraffin-embedded specimens, and the glass slides used had been pretreated with poly-L-lysine hydrobromide (Sigma Chemicals), followed by drying at 65°C for 12 hours. Sections were dewaxed in xylene, hydrated in a graded series of ethanol, washed with distilled water, boiled for 10 minutes in 5% urea in a microwave oven, and cooled for 30 minutes before incubation with HCD30C2 or Ber-H2. A Histofine kit (Nichirei, Tokyo, Japan) was used for color reaction for alkaline phosphatase, according to the manufacturer’s instructions. The following reagents (all from Dako Japan) were used in this study: biotinylated swine anti-rabbit IgG, biotinylated goat anti-mouse IgG, and alkaline phosphatase-conjugated streptoavidine.
Cell Labeling and Immunoprecipitation
COS-7 cells were transiently transfected with pME-CD30v22 or an empty vector, and after 40 hours, about 2 x 106 cells in semiconfluent culture were washed with PBS (-) and cultured in phosphate-free Dulbecco’s minimum essential medium for 18 hours before the addition of 1 mCi/ml of [32P]orthophosphate (carrier-free; ICN Biomedicals, Costa Mesa, CA). The cells were then incubated for a further 5–6 hours before immunoprecipitation. Cells were lysed as described above, and the cell lysates were first incubated with protein G Sepharose (Pharmacia) at 4°C for 60 minutes. After centrifugation, supernatants were incubated with 2 µg of HCD30C2 at 4°C for 60 minutes and then incubated with protein G Sepharose (Pharmacia) at 4°C for 60 minutes. For competition studies, 2 µg of HCD30C2 was preincubated with 10 µg of the antigen peptide for 30 minutes on ice. The immune complexes absorbed to protein G Sepharose were washed with modified RIPA buffer. The samples were boiled with Laemmli’s buffer for 5 minutes and subjected to 15% SDS-PAGE followed by autoradiography.
In Vivo Binding Assay
Transient cotransfection and coimmunoprecipitation analyses were made using 293T cells. Expression vectors for mouse TRAF2 and TRAF5 tagged with FLAG were as described.17,21,22 Cells were cotransfected with 1 µg of an expression plasmid for human CD30v or CD30, and 1 µg of expression vectors for FLAG-tagged TRAF proteins, using Lipofectin reagent (GIBCO BRL, Gaithersburg, MD). The cells were lysed in HNE buffer (50 mM HEPES, pH 7.2, 0.1% Nonidet-P40, 100 mmol/L NaCl, 10% glycerol, 2 mmol/L EDTA, 1 mmol/L PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 mg/ml pepstatin), the lysates were first immunoprecipitated with HCD30C2 or Ber-H2, and the immunoprecipitates were analyzed by immunoblotting with anti-FLAG M2 antibody (Eastman Kodak) and peroxidase-conjugated sheep anti-mouse immunoglobulin antibody, using an ECL kit (Amersham).
Statistical Analysis
The association between the expression of CD30v and that of CD30L
in AMLs and LPDs was statistically assessed by 
2
test.33
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of CD30v transcripts in malignant cells of myeloid and
lymphoid origin was investigated by RT-PCR. Because organization of the
CD30 gene has yet to be defined, primer pairs cannot be designed to
locate in different exons for discrimination between products from cDNA
and those from genomic DNA. Therefore, we prepared two sets of primer
pairs, whose nucleotide sequences were selected by choosing, in both
cases, a sequence within the 5' unique CD30v sequence (nucleotide
position 27–49) for forward primers, and reverse primers localized
either upstream of the putative translation initiation codon
(nucleotide position 228–247, Figure 1A
,
primer pair A) or less than 100 bp upstream of the stop codon
(nucleotide position 598–575, Figure 1A
, primer pair B).
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(lanes 1 and 2), the same PCR
products were obtained by amplifying with CD30v-specific primers A
cDNAs that have been synthesized from RNAs treated with RNase-free
DNase I or not. On the other hand, PCR products were never observed
with the same primer pair when either non-reverse-transcribed RNA or
control genomic DNA from peripheral blood mononuclear cells was used as
the template (Figure 1B
, lanes 3 and 4). Similar results were obtained
with primer pair B, which amplifies the larger CD30v fragment (sense
nucleotide position 27–49; antisense nucleotide position 598–575;
data not shown).
In addition, further experiments were designed to rule out the
possibility that primer pairs specific for CD30v might cross-amplify
CD30 transcripts. When cDNAs from CD30-expressing cell
lines1-6,22
were used, amplification with both CD30v
primer pairs failed to produce specific PCR products (Figure 1C
, upper
panel, and data not shown). Conversely, CD30 expression in
CD30-positive cell lines was confirmed by RT-PCR (Figure 1C
, lower
panel), by using primer pairs specific for the full-length form of CD30
(Figure 1A
, primer pair C). On the other hand, the same primer pair did
not give rise to specific amplified products when TPA-treated HL-60
cells were tested (Figure 1C
, lower panel). Taken together, these
results indicated that RT-PCR products obtained with our CD30v primer
pairs specifically derived from CD30v cDNA templates and not from
genomic DNA or full-length CD30 cDNA molecules.
Expression of CD30v mRNA in Malignant Cells of Myeloid Origin
Expression of CD30v-specific transcripts was investigated in blast
cells purified from 72 AML cases, and 11 cases of MBC evolved from
previous chronic myeloproliferative disorders (CMDs). As shown in Table 1
and Figure 1D, a
broad expression of
CD30v mRNA was found in these malignancies. In detail, significant
amounts of CD30v mRNA were detected in 52 of 72 (72.2%) AMLs and 7 of
11 (63%) MBC of previous CMDs (Table 1)
. Although not reaching a
statistical significance, a higher frequency of CD30v expression
(81.8%) was detected in monocytic and myelomonocytic subtypes of AMLs
(FAB M4 and M5), as compared to immature-intermediate AML phenotypes
(FAB M0, M1, and M2), in which CD30v-positive samples accounted for
66.7% of cases (Table 1)
. Even though RT-PCR procedures were not
specifically conditioned to be quantitative, leukemic samples from
promyelocytic (FAB M3) and monocytic-oriented leukemias (FAB M4 and M5)
appeared to express higher levels of CD30v transcripts overall than did
those from M0, M1, and M2 AML subtypes (Figure 1D)
. With regard to the
expression of long (CD30vL) and short (CD30vS)
forms of CD30v mRNA,22
almost all cases (50 of 52) showed
expression of either the long form (CD30vL) alone (30
cases) or the long form in association with the short form
(CD30vS) (20 cases), the latter being expressed in the
absence of CD30vL only in two M5 AML samples (Table 1)
.
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CD30v expression was also surveyed in 90 cases of B-cell tumors
and 30 cases of T-cell tumors. As was the case for myeloid cells, 70%
of B-cell malignancies (63 of 90) expressed CD30v transcripts, and
again the great majority (60 of 63) showed either expression of the
CD30vL isoform alone (33 cases) or coexpression of
CD30vL and CD30vS (27 cases; Table 2
and Figure 1E
). About 50–60% of
CD30v-expressing cases were observed in B-lineage ALLs, high-grade
B-NHLs, B-PLL, HCL, and low-grade B-NHL. Interestingly, a very high
rate of CD30v expression was observed in B-CLLs (26 of 27) and in
immunopurified BB-4+ malignant plasma cells (6 of 6). In contrast, less
than 25% of T-cell malignancies, including ATL caused by HTLV-1,
expressed CD30v transcripts.
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Because expression of a given gene is not always associated with the actual detection of its protein product,34 we attempted to survey the expression of CD30v protein in malignant cells of myeloid and lymphoid origin. For this, we generated a rabbit polyclonal antibody, termed HCD30C2, using as the immunogen the amino-terminal peptide of the CD30v. Because this amino acid sequence is shared by CD30 and CD30v proteins (amino acids 464–472 and 1–9, respectively), we first tested the specificity of HCD30C2 reactivity. To do this, immunoprecipitation and immunoblotting experiments were done using COS-7 cells transfected with expression vectors specific for CD30 or CD30v.
In agreement with previously reported data,22
HCD30C2
antibodies, while failing to detect CD30 protein in CD30-transfected
COS-7 cells (Figure 2A
b), did recognize
two closely migrating bands with an apparent molecular mass of about 25
kd, but only in cells transfected with CD30v (Figure 2Ab
). Some
additional bands in most samples had a molecular mass slightly higher
than that of bands identified as CD30v-specific (asterisk in Figure 2Ab
), and this was considered a nonspecific reactivity of HCD30C2
antibodies with some unidentified components of cellular lysates.
Conversely, the anti-CD30 monoclonal antibody Ber-H2 strongly reacted
with COS-7 cells transfected with the full-length form of CD30, being
totally unreactive with cells transfected with CD30v (Figure 2Aa
).
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. Taken together, these results demonstrated
that HCD30C2 reacts to CD30v protein but not to CD30 protein.
In a subsequent series of experiments, we examined the potential of
HCD30C2 to recognize CD30v protein in viable cells fixed by
paraformaldehyde and in formalin-fixed and paraffin-embedded samples.
We first asked if CD30v protein in paraformaldehyde-fixed cells could
be detected by indirect immunofluorescence cytochemistry. Cytoplasmic
reactivity was clearly observed in CD30v-transfected COS-7 cells but
not in CD30-transfected COS-7 cells by HCD30C2 (Figure 3A, a and c)
. Conversely, Ber-H2
specifically reacted to COS-7 cells transfected with CD30 but not to
those transfected with CD30v, as expected (Figure 3A, b and d)
.
Consistently, HCD30C2 reacted to cytoplasmic antigen only in the
TPA-treated HL-60 cells, not in untreated HL-60 cells (Figure 3B, a and b)
. Similarly, HCD30C2 antibodies recognized both recombinant and
native CD30v protein, as expressed in CD30v-transfected COS-7 cells and
TPA-treated HL-60 cells, respectively, in formalin-fixed and
paraffin-embedded preparations (data not shown).
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Expression of CD30v Protein in Acute Leukemia Cells of Myeloid and Lymphoid Origin
Using the CD30v-specific polyclonal antibody HCD30C2, we next
surveyed the expression of CD30v protein in 35 BM samples from
patients affected by acute leukemias of myeloid or lymphoid origin, by
indirect immunofluorescence or immunohistochemistry (Figure 3B, c and d
, Figure 3C
, and Table 3
). Indirect
immunofluorescence studies of paraformaldehyde-fixed bone marrow
samples from six AML patients revealed CD30v protein in the cytoplasm
in two of six samples (Figure 3B, c and d
, and Table 3
). In addition,
immunohistochemical studies with HCD30C2 revealed a significant
expression of CD30v in 8 of 19 AML cases and 2 of 10 ALLs.
As shown in Figure 3C
, cytoplasmic expression of CD30v was
detectable in leukemic blasts, whereas no staining was observed in
normal bone marrow specimens. In particular, whereas in
some cases CD30v-expressing blasts were seen as loose clusters
interspersed among a population of CD30v-negative cells (Figure 3Ca
),
in other samples expression of CD30v was characterized by a disperse
granular staining of the cytoplasm, in the majority of blasts
(Figure 3C, b and c)
. Thus immunological screening for CD30v
protein expression in acute leukemia samples revealed
CD30v-expressing cases in 10 of 25 AML samples and 2 of 10 ALL
samples (Table 3)
. Finally, in agreement with reported
data,10
all leukemic samples lacked expression of CD30
antigens, as investigated by immunostaining with anti-CD30 mAb Ber-H2
antibody (data not shown).
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Screening by RT-PCR with a specific primer pair for CD30v and
immunostaining with HCD30C2 showed frequent expression of CD30v in
human malignant myeloid and lymphoid neoplasms. For further
confirmation, we examined the parallel expression of CD30v transcripts
and protein in four AML cases (M1, M2, M4, and M5 subtypes) and two HCL
cases by Northern blotting and immunoblotting. As shown in Figure 4A
, Northern blot analysis revealed a
2.3-kb signal corresponding to CD30v transcripts22
in two
of four AMLs (M4 and M5 subtypes) and in the HCL cases tested, which
were all documented as CD30v-positive by RT-PCR. Consistently, no
specific bands were detected in two AML cases (M1 and M2 subtypes) that
lacked expression of CD30v mRNA by RT-PCR. Finally, a specific CD30v
transcript was also clearly detected in TPA-treated HL-60, but not in
HL-60 cells (Figure 4A)
, used as positive and negative controls,
respectively.22
Immunoblotting experiments with HCD30C2
antibodies confirmed the presence of a 25-kd CD30v-specific band in
those cases, ie, two AMLs, two HCLs, and TPA-treated HL-60, expressing
significant amount of CD30v transcripts by RT-PCR and Northern
blotting, but not in a CD30v mRNA-negative case (M1 or unstimulated
HL-60) (Figure 4B)
.
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Based on the data we accumulated, CD30v was expressed frequently
in malignant cells of myeloid and lymphoid origin. Because we earlier
reported the frequent expression of CD30L in human hematopoietic
malignancies,10
it was of interest to analyze the
parallel expression of CD30v and CD30L transcripts in tumor cells from
42 AMLs and 51 LPDs in our series. In agreement with earlier
findings,10
a high rate of CD30L-expressing cases was
observed both in myeloid and lymphoid malignancies (Figure 5)
, as opposed to CD30 mRNA that was
detected only in 2 of 42 AML cases and 4 of 51 LPDs (not shown). A
statistically significant correlation was found between the expression
of CD30L mRNA and the presence on malignant cells of specific
transcripts for CD30v. As shown in Figure 5
, 2
analysis
performed by combining results of CD30v and CD30L expression in AMLs
and LPDs revealed a P value of 0.02 and an odds ratio of
3.22, thus indicating a more than threefold higher possibility that
CD30v-expressing cells coexpress CD30L, as compared to tumor cells
lacking CD30v expression.
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We reported that signals emanating from CD30 can activate NF-B
through interactions of TRAF2 and TRAF5 proteins with specific domains
localized in the C-terminal region of CD30.17,21
Because
CD30v protein retains these TRAF-binding domains and can activate
NF-B, as the authentic CD30 protein,22
interactions
between CD30v and TRAF proteins were examined in coimmunoprecipitation
experiments, using expression vectors for CD30v or CD30 and FLAG-tagged
TRAF2 or TRAF5 proteins (Figure 6)
. When
the CD30v was expressed along with TRAF2 or TRAF5 in 293T cells,
coimmunoprecipitation of CD30v with TRAF2 or TRAF5 was clearly
demonstrated (Figure 6)
, indicating in vivo interactions
between CD30v and TRAF proteins. Because we observed that the
cytoplasmic region of CD30 interacts with the corresponding
CD30v (unpublished observation), these results suggest that
aggregation of CD30v protein itself may result in NF-B activation by
recruiting TRAF proteins.
|
,
blast cells from all seven cases analyzed expressed significant amounts
of transcripts for TRAF1, TRAF2, TRAF3, and TRAF5 proteins, except for
two cases lacking TRAF1 expression (Figure 7b)
. Although the expression
of transcripts specific for TRAF proteins was not exclusively confined
to CD30v-positive cases (the expression was also found in
CD30v-negative malignancies; data not shown), our present results do
indicate that CD30v may interact in vivo with the
NF-B-activating proteins TRAF2 and TRAF5, the specific transcripts
of which are significantly expressed in CD30v-expressing leukemic
blasts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activating signals. Interestingly,
such a frequent expression of CD30v seems to be exclusively confined to
human malignant myeloid and lymphoid neoplasms, because a survey of
CD30v expression in a wide panel of cell lines originating from
extrahematopoietic tumors overall failed to detect specific CD30v
transcripts (V. Gattei et al, unpublished observations). Screening of
CD30v transcripts by RT-PCR in 83 myeloid and 120 lymphoid malignancies
demonstrated a frequent expression of CD30v—71% and 58% in myeloid
and lymphoid malignancies, respectively. In the myeloid series, we
observed that CD30v is more frequently expressed in cases classified in
FAB M4 and M5 than in those in M0, M1, and M2. Frequent and higher
levels of CD30v expression in promyelocytic and monocyte-oriented
leukemias are in accord with our earlier observations that CD30v is
expressed in alveolar macrophages.22
In the lymphoid
series, B-lineage tumors showed a higher rate of CD30v expression than
did T-lineage tumors (70% versus 23%), with almost 100%
of CD30v-expressing cases found in B-CLL and multiple myeloma. It
remains to be investigated whether CD30v expression is related to
differentiation or transformation of B cells.
Detection of both CD30v transcripts and protein by combined analysis,
using Northern blot and immunoblot analyses for cases positive by
RT-PCR, provided further evidence for expression of CD30v in human
malignant myeloid and lymphoid neoplasms. These results also provide
strong support for the notion that results from RT-PCR represent
expression of CD30v, at a significant level (Figure 4)
.
We also noted the presence of the long (CD30vL) and short
(CD30vS) forms of CD30v transcripts, which are probably
generated by alternative splicing.22
Both forms of CD30v
transcripts were found in malignant cells of myeloid and lymphoid
origin (Tables 1 and 2)
. However, in 123 of 129 CD30v-expressing cases,
CD30vL is expressed alone or in association with
CD30vS, whereas CD30vS expression in the
absence of CD30vL was found in only six cases (Tables 1 and 2)
. Alternatively, CD30vS was detected in 60 of 129
CD30v-positive cases. It was not clear whether the lower detection rate
of CD30vS is due to preferential expression of
CD30vL, to lower levels of CD30vS transcripts,
or to differences in the amplification efficiency in RT-PCR. The lower
level of expression of the shorter transcripts is favored because the
magnitude of amplification of the shorter transcripts was always lower
than that of the larger one when both transcripts were detected.
An interesting correlation between expressions of CD30v and CD30L
became evident in analyses of 42 AMLs and 51 LPDs (Figure 5)
.
Coexpression of these molecules may result from a common regulatory
mechanism of transcription. Although the molecular mechanisms of
transcriptional regulation have yet to be determined, CD30L expression
in lymphocytic cell lines and peripheral blood monocytes and T cells
may be induced by activating agents such as CD27 ligand, phorbol ester,
and Staphylococcus aureus Cowan antigen.35
The
regulation of CD30v expression has yet to be characterized, and
mechanisms explaining the coexpression between CD30v and CD30L remain
to be determined. However, our data are of some interest because the
simultaneous expression of CD30v and CD30L has been noted, in the
absence of CD30, in normal peripheral blood T cells from patients
affected by helminthic infections.36
Moreover, the
parallel up-regulation of CD30v- and CD30L-specific transcripts was
also observed after in vitro activation of peripheral blood
mononuclear cells from healthy donors by helminthic
antigens.36
Finally, high levels of constitutive CD30v and
CD30L mRNA and protein were noted in normal peripheral blood
neutrophils (Gattei et al, unpublished observation).10
Interestingly, reverse signaling via CD30L was observed in freshly
isolated neutrophils and T cells suboptimally stimulated by anti-CD3,
leading to production of interleukin-8 and a rapid oxidative burst, or
to an increased metabolic activity, proliferation, and production of
interleukin-6, respectively.11
It will be of interest to
determine whether CD30v plays a role as a cytoplasmic signal transducer
in processes of reverse signaling through CD30L, in these cell types,
by interacting with the cytoplasmic N-terminus of CD30L. Experiments
designed to test this possibility are now under way in our laboratory.
The rabbit antibody HCD30C2 raised against the N-terminal peptide of
CD30v protein discriminated CD30v from the authentic CD30 in immunoblot
analysis and immunoprecipitation. In addition, as shown in
immunofluorescence studies, HCD30C2 reacted specifically with
cytoplasmic antigens in TPA-stimulated HL-60 and COS-7 cells that
express CD30v protein, indicating minimal or no reactivity of HCD30C2
with other antigens in the cells. Although the sequence of the antigen
peptide of this antibody is shared by CD30v and CD30, this selective
reactivity probably depends on the positive charge of the -amino
group in the N-terminus of the CD30v protein, which is the only
difference compared with CD30. Similarly, antibodies that selectively
recognize the forms of protein kinase C have been developed with
the use of peptide immunogens.37,38
As observed in immunoblot experiments, HCD30C2 seems to recognize two closely migrating bands with an apparent molecular mass of about 25 kd. As previously suggested,22 these two bands may be caused by an alternative translation initiation from two closely located ATG codons or may be the result of an unidentified posttranslational modification of the CD30v protein.
In addition to the CD30v protein, HCD30C2 also reacted nonspecifically
with a band in immunoblot analysis. A similar but less evident
nonspecific band was also observed in immunoprecipitation experiments.
Pretreatment of the antibody with the immunogen resulted in a
significant reduction in intensity of the band, suggesting that this
protein has an epitope recognized by HCD30C2 (Figure 2B
and data not
shown). Notwithstanding these results, reactivity of HCD30C2 appeared
specific in immunofluorescence and immunohistochemistry, as mentioned
above. These observations provide the basis for using HCD30C2 to detect
CD30v expression in tissues.
The detection rate of CD30v expression in myeloid (10 of 25) and
lymphoid (2 of 10) leukemia samples by immunohistochemistry and
immunofluorescence cytochemistry was lower than that in RT-PCR analyses
(Table 3)
; the difference is probably the result of a lower sensitivity
of immunological detection compared to that of RT-PCR, because it is
more frequently detected in AML than in ALL. In most CD30v-expressing
cases, CD30v-positive cells were noted among CD30v-negative blasts. It
remains to be determined whether this finding actually reflects a
smaller number of CD30v-expressing cells among CD30v-negative blasts or
is the result of selective detection of highly expressing cells among
many CD30v-expressing blasts. These possibilities can be defined by
studies with a more sensitive method of immunohistochemistry and/or a
sensitive detection of CD30v transcripts, using in situ
hybridization. Studies of the function and regulation of the cryptic
promoter for CD30v transcripts will also provide clues to how CD30v
expression is regulated in leukemia cells.
Demonstration of interaction between CD30v and TRAF proteins and
expression of transcripts for TRAF proteins provide the basis for
assuming functional roles of CD30v in leukemia cells. We and other
investigators found that NF-B activation is mediated by interaction
of TRAF2 and TRAF5 with the C-terminal region of the CD30 cytoplasmic
domain,15-20
which is retained in CD30v. Thus NF-B
activation by CD30v is likely to be mediated by interaction with these
TRAF proteins.
Although NF-B activation in CD30v-expressing leukemia cells, as
compared with CD30v-negative ones, was not examined directly, the
expression of TRAF-specific transcripts by malignant cells of
CD30v-positive cases strongly favors the view that a CD30v-mediated
NF-B activation in CD30v-positive cells may play a role in the
growth and/or differentiation of these cells.
In summary, we demonstrated the frequent expression of CD30v as well as
transcripts for CD30v-associated signal transducer proteins in leukemic
blasts, a finding that suggests roles for CD30v in growth and/or
differentiation of these cells. Further studies of the mechanism
underlying selective activation of the cryptic promoter for CD30v and
direct examination of NF-B activation in CD30v-positive cells are
expected to provide insight into the biological effects caused by CD30v
protein in CD30v-positive cells.
|
Acknowledgements |
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|
Footnotes |
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Supported in part by a Grant in Aid for Scientific Research and a Grant in Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture, Japan (to T. W.); the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to R. H.); and grants from the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Milan, Italy; the Associazione Italiana contro le Leucemie (A.I.L.) "Trenta ore per la vita," Italy; and the Ministero della Sanità, Ricerca Finalizzata I.R.C.C.S., Rome, Italy.
R. H. and V. G. contributed equally to this work.
Accepted for publication August 24, 1999.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation. Int Immunol 1998, 10:203-210. Biochim Biophys Acta 1995, 1269:253-259[Medline]
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