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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2443-2451
By
From Georgetown University Medical Center, Washington, DC; and Oregon
Health Sciences University, Portland, OR.
Although Hodgkin's disease is highly responsive to treatments that
cause apoptosis, it remains resistant to the physiological mechanisms
intended to cause cell death. Presumably, the Reed-Sternberg cell
defies endogenous apoptosis, persists, accumulates, and manifests the
malignant disorder seen clinically. The Reed-Sternberg cell expresses
several members of the tumor necrosis factor receptor superfamily. This
family of receptors is involved in both activation and proliferation of
cells, as well as either protection from or initiation of apoptosis in
cells expressing these surface proteins. Signals from these receptors
affect transcription. We reasoned that the activation state and
resistance to apoptosis of Reed-Sternberg cells might be attributable
to dysregulation of genes controling these processes. To determine gene
expression by Reed-Sternberg cells, we developed a method of
micromanipulation, global reverse transcription, and the reverse
transcription-polymerase chain reaction and applied it to 51 single
Reed-Sternberg cells and their variants from six cases of Hodgkin's
disease. This report analyzes the gene expression of bcl-xs,
bcl-xl, bax-
THE ORIGIN of the Reed-Sternberg cell and
the biological mechanisms of its growth, regulation, and death remain
largely unstudied. This is a consequence of the rarity of the
Reed-Sternberg (RS) cell in involved tissues and the difficulty in
establishing long-term cultures of primary cells. Despite this,
Hodgkin's disease (HD) is often treatable by chemotherapy and
radiation therapy.1 The effectiveness of therapy is
presumed to be mediated by induction of apoptosis, a form of programmed
cell death.2-4 Even histologically, a low level of
spontaneous apoptosis in RS cells is suggested by certain karyorrhectic
"mummified" cells with condensed nuclei seen in tissue
sections.5 Their sensitivity to therapy and endogenous cell
death argues that neoplastic cells of HD retain the capacity to undergo
apoptosis. The physiological apoptosis, however, is not sufficient to
keep the disease in check, as HD spreads inexorably when left
untreated. Still other cases resist therapy. Here, we examined cellular
pathways in primary RS cells to better understand the molecular
mechanisms that cause the RS cell to survive.
Patterns of cell growth, survival, and programmed death (apoptosis) are
central to the development and homeostasis of the organism;
inappropriate downregulation of apoptosis appears to be a common theme
in neoplasia, including non-Hodgkin's lymphoma (NHL). One family of
apoptosis-regulating proteins, which includes Bcl-2, Bcl-xl, Bcl-xs,
Bax, Bak, and Bad, is dysregulated in NHL.6-8 Bcl-2 and
Bcl-xl provide protection from cell death, whereas Bcl-xs, Bax, Bak,
and Bad promote death of the cell by apoptosis. Ratios of death to
anti-death proteins within the cell are critical, as anti-death
proteins probably function by binding to and inactivating apoptosis
proteins. In HD, immunohistochemical studies have focused on Bcl-2,
Bcl-x, and Bax, showing that all three proteins are expressed by more
than 50% of the RS cells, thereby suggesting a mechanism for both in
vivo survival and responsiveness to therapy.9,10
The tumor necrosis factor receptor (TNFR) superfamily of cell-surface
molecules elicits a panel of responses from proliferation to apoptosic
cell death in cells of the immune system and are involved in regulating
the BCL family proteins. The RS cell expresses many of these proteins
including TNFRs 1 and 2, CD30, CD40, and Fas (CD95).11,12
This group of receptors may be divided functionally into those that
induce apoptosis when bound by their ligands and those that promote
survival and proliferation. TNFR1 and Fas signal through a
cytoplasmic "death domain" bound by the intracellular molecules
TRADD (TNFR-associated death domain) and FADD (Fas-associated death
domain), respectively.13,14 This pathway may involve the
death-promoting Bax protein. In the case of TNFR2, CD30,
and CD40, a TRAF (TNFR-associated factor)-binding domain binds one or
more of four TRAFs.15-19 Interestingly, the LMP 1 protein
of Ebstein-Barr virus (EBV), whose products are present in the RS cells
in more than 70% of HD cases,20 also contains a TRAF
domain and uses the TRAFs in some aspects of its
signaling.17 CD40 and LMP 1 upregulate the antideath
proteins Bcl-xL and Bcl-2, respectively, and may thus promote
survival.21,22
Cell-surface signaling of apoptosis promoted by engagement of Fas with
its ligand (FasL) may also involve another family of proteins
structurally similar to interleukin-1 Loss of function of either Fas or FasL leads to unregulated
proliferation of lymphocytes, as evidenced by lpr mice, which lack Fas
as a result of a germline mutation of the gene, and gld mice, which
lack FasL.29-33 Similarly, a germline defect in Fas in
humans can cause an autoimmune lymphoproliferative syndrome (ALS).34 HD is thought to be a lymphoproliferation with a
profound immune response and might be a manifestation of an ALS.
However, somatic mutations of these genes have not been examined in HD.
Both TNFR1 and TNFR2 and their ligands, the TNFs, are
expressed by RS cells.11 Survival and activation promoted
by TNFRs may be mediated through the TRAFs. TRAFs 1, 2, and 5 can
activate the transcription factor nuclear factor The repertoire of expressed genes determines the balance between cell
growth, activation state, and apoptotic fate. RS cells may be apoptotic
and express members of the TNFR superfamily, but for most of the cell
death and TNFR-associated proteins no antibody is suitable for
immunohistochemical detection. Here, we specifically examine gene
expression at the level of transcription in single RS cells from
primary tissues. RS cells appear to have a defective germinal center
B-cell phenotype based on their rearranged but nonproductively
hypermutated IgH genes36 and a very limited growth
fraction, as suggested by DNA content analysis.37 Their persistence in tissue must be partly attributable to regulated cell
death. We have begun to test the hypothesis that RS cells and their
variants may persist in the untreated state as a result of dysregulated
expression of apoptosis-controlling genes. We have used a series of
single RS cell 3 Cells and Tissues
B cells.
Fresh tissue was obtained from tonsillectomy specimens, minced in RPMI
medium with penicillin (100 U/mL) and streptomycin (100 µg/mL) and
passed through a sterile wire mesh to create a single cell suspension.
Mononuclear cells were partially purified by centrifugation over
Ficoll. B cells were purified from this population by rosette depletion
of T cells with sheep red blood cells as described.39 The
B-cell population was 76% pure, as confirmed by the presence of
surface antigens CD19 and CD20 and the absence of markers CD2 and CD7
as evaluated by flow cytometry.
T cells.
Leukopheresis-enriched peripheral blood was obtained from the American
Red Cross (Baltimore, MD). Mononuclear cells were partially purified by
centrifugation over Ficoll. 2 × 109 cells were
resuspended in 10 mL sterile RPMI medium and passed over a nylon wool
column. After 1-hour incubation at 37°C, nonadherent cells were
eluted with 75 mL RPMI at 37°C. The resulting T-cell population was
82% pure, as confirmed by the presence of surface antigens CD2 and CD7
and the absence of CD19 and CD20 as evaluated by flow cytometry.
Cell lines.
KMH2 was kindly provided by Dr Hiroshi Kamesaki (Nagoya,
Japan), and L428 was provided by Dr V. Diehl (Cologne, Germany). Jurkat
and Raji cell lines were obtained from American Type Culture Collection
(ATCC, Rockville, MD). All lines were maintained in RPMI 1640 containing 10% fetal calf serum (FCS) (GIBCO-BRL, Grand Island, NY)
and antibiotics in a humidified atmosphere with 5% CO2.
mRNA and cDNA
Single-Cell RT-PCR
Primers The following primer pairs were used. For each set, the upper strand (5 ) primer is listed first:
Immunohistochemistry Tissue sections were deparaffinized and stained with a monoclonal anti-Bcl-2 antibody (DAKO, Carpinteria, CA), anti-Bax monoclonal antibody (Pharmingen, San Diego, CA) or anti-Bax polyclonal antibody (kindly provided by Dr John C. Reed, La Jolla, CA) using an indirect avidin-biotin-peroxidase complex (ABC).51 Positive controls were performed with a single block containing multiple tissues, including placenta, lymph node, spleen, large cell lymphoma, and liver.
Optimization of PCR Conditions PCR conditions for each set of primers were optimized using an appropriate positive control cDNA known to express the gene of interest (see Materials and Methods). In addition, the positive control cDNA was included in each set of reactions with the single-cell cDNAs to verify reaction conditions. In each round of PCR, the positive control cDNA was consistently positive, and the negative (no template) controls were negative (data not shown), confirming the efficacy and validity of each round of the PCR.Validation and Quantification of the Expanded Single-Cell cDNA Libraries To test the relative quality of the reamplified cDNA libraries, each expanded set of single cell cDNAs was analyzed for the expression of a ubiquitously expressed gene and for the absence of genomic DNA. First, each was screened for -actin using primers that lie within the last
exon of the gene. All the cDNA libraries generated for this study
contained -actin titratable out to 1 : 5,000 dilution of the
reamplified sample, indicating that the mRNA of a randomly selected,
constitutively expressed gene could be consistently detected in all
expanded cDNA libraries (Fig 1). To confirm
that the cDNA libraries contained only expressed sequences, they were
screened for the presence of genomic DNA. For this purpose, a primer
was derived from the untranscribed sequence of intron 3 of the
2-microglobulin gene26 and paired with a
primer complementary to a sequence in a downstream exon of this gene. A
PCR product with this pair of primers would be derived only from
unspliced DNA, and thus would indicate genomic contamination of the
library. Forty-two of 51 cDNA libraries tested in this manner gave no
PCR product with this pair of primers and were included in the
analysis.
RT-PCR Analysis of Apoptosis-Regulating Genes Although all the selected apoptosis-associated genes were found to be expressed in single RS cells and their variants, their frequency of detection was highly variable. The transcript for bcl-xl was detected in one or more of the RS cells and their variants in four of six cases of HD studied. The transcripts for bax- or bax-, however,
were found in one and five, respectively, of the 42 RS cells and their
variants analyzed. Another member of the bcl-2 family,
bak, was found in 13 of 42 RS cells analyzed (Table
1).
RT-PCR Analysis of TNFR Signaling Proteins Ligands.
TNF-
Receptors. Both TNFR1 and TNFR2 were detected in all cases of HD but at low (10 cells and 5 cells, respectively) frequency. Fas (CD95) gene expression was frequent (24 of 42 RS cells) (Fig 2; Table 1), but FasL mRNA was rarely detected.
Cytoplasmic mediators. TRAF1, 2, and 3 were detected in all cases of HD examined. TRAF2 and 3 were present evenly throughout the cases, while TRAF1 appeared to be expressed either highly (more than 50% of cells) in four of six or rarely or not at all (two of six cases). cIAP2 transcript was found in 57% of RS cells, more than twice the frequency of TRADD transcript. FADD transcript was detected very frequently (79% of cells) in the case of LP HD but was rare to absent in the NS and MC cases analyzed. ICE transcript was detected in one or more cells in every case of HD analyzed (Table 1). Immunohistochemical Detection of Bcl-2 and Bax Protein in Selected Cases Cases 1, 3, 5, and 7 were analyzed by immunohistochemistry for Bcl-2 protein expression. Bcl-2 protein was found to be expressed strongly in cases 3 and 7, moderately in case 5, and weakly in case 1. Case 2, 6, and 7 were analyzed with both a polyclonal antibody and a monoclonal antibody directed against the Bax protein. All cases were negative for Bax, using the monoclonal antibody, whereas the polyclonal antibody reacted with RS cells strongly in case 6, moderately in case 7, and faintly in case 2. Lymophocytes displayed negative staining in all cases with both antibodies (data not shown).Clinical Data All patients from whom cells were used in this study achieved complete remission for at least three years (Table 3). Patient number 7 relapsed at 3 years and subsequently demised despite treatment.
HD Cell Lines The Hodgin-derived cell lines KMH2 and L428 were tested by RT-PCR for the expression of each gene that was assayed in single, primary RS cells (data not shown). KMH2 was found to express all the genes in this study except FasL. L428 expressed transcripts for all except FasL and TNFR1.
The single-cell strategy applied here overcomes the previous obstacles in gene expression analysis of RS cells by examining the mRNA of isolated living cells, uncontaminated by surrounding host cells. Several single cells in each case were evaluated to provide a more accurate picture of gene expression because it is likely that expression of genes, even those constitutively expressed, may vary among individual cells. Because of this, the level of expression may be extrapolated from the number of positive or negative cells in a population. The data from these experiments were evaluated on this basis. TNFR Family Signaling May Favor Survival RS cells and their variants are known to express members of the TNFR superfamily. For example, CD30 and CD40 are abundantly expressed by RS cells in most HD cases, as shown by immunohistochemistry.11 TNFRs 1 and 2 were shown previously to be infrequently detected by immunohistochemistry, and this was corroborated in this study by RT-PCR. In the present study, RS cells were found to express mRNA of TNF- and -, the ligands of these receptors, which can act in an
autocrine, paracrine, or endocrine manner in HD. The intracellular
pathways initiated by these four TNFRs may be directed toward cell
survival. The downstream signals of these receptors, however, have not
been previously examined in primary RS cells, and appropriate
immunohistochemical reagents are not available. Here we have
specifically looked at cytoplasmic mediators of the TNFR family by
single-cell cDNA analysis.
Relative Levels of Apoptosis Family Members The findings presented in this report suggest that Reed-Sternberg cells and their variants suppress apoptosis by the regulation of genes involved in a cell death pathway. For example, Bax promotes cell death in mammalian cells when its level is high relative to that of Bcl-2 or Bcl-xl,43 and there was infrequent expression in single RS cells of both bax- and
bax-, which are known to induce apoptosis when
abundant. Interestingly, the transcript for the anti-death gene
bcl-xl was detected more frequently in RS cells than those for
the death promoting genes bax- or
bax-, suggesting that the cells may be
protected from death by Bcl-xl, at amounts capable of binding all
available Bax. Bcl-xs may function by heterodimerizing with Bcl-xl,
thereby displacing Bax and promoting cell death44 and its
expression may play a role in the responsiveness of HD to chemo- and
radiation therapy. CD40 upregulates the expression of Bcl-xl and,
therefore, CD40 signaling may be responsible for protecting the RS cell
by this pathway. The anti-apoptosis protein Bcl-2 was detected by IHC
in all cases tested and may function in a similar manner.
Receptors for T-Lymphocyte-Mediated Cell Death Are Present in RS Cells Fas, known to be expressed on the surface of RS cells by IHC,12 was detected here at high frequency by RT-PCR. RS cells are often surrounded in vivo by adherent, activated T cells capable of expressing the ligand for Fas. When activated by its ligand, Fas can induce apoptosis in lymphocytes.60-62 Cytotoxic T lymphocytes are known to express FasL and are thought to mediate killing through activation of Fas on target cells63 by activation of downstream ICE-like cysteine proteases or "caspases."28 Overexpression of ICE itself is capable of inducing apoptosis.24 Both Fas and ICE were detected at the mRNA level in RS cells. However, Bcl-2 and Bcl-xl prevent a step in the Fas pathway that occurs before caspase activation. Since RS cells and their variants survive in vivo, the caspases in these cells may be in an inactive form. The Fas/FasL pathway may be active in some RS cells, because occasional evidence of presumably apoptotic cells with karyorrhectic nuclei is seen histologically. This mode of killing, however, must be insufficient to cause the complete elimination of RS cells and to prevent the spread of disease seen in untreated patients.Apoptosis Genes Allow Alternate Pathways to Cell Death Because HD is very sensitive to treatment, alternative pathways of apoptosis may be intact. In fact, all patients from whom cells were used in this study achieved complete remission for at least 3 years (Table 3). The presence of bax, bak, and ICE transcripts in RS cells leaves the opportunity for apoptosis signaling through these pathways. These cytoplasmic effectors may be upregulated or activated in RS cells exposed to chemotherapeutic agents or irradiation, which presumably act by inducing apoptosis in the tumor cells. Chemotherapy with Adriamycin upregulates FasL expression on certain T-cell leukemia cells and kills the leukemia by inducing Fas-mediated apoptosis.65 This mechanism may also play a role in Adriamycin-treated HD, enhancing the ability of the T cells to kill the RS cell through the Fas pathway.53 It would be intriguing to determine the effects of treatment on expression of these and other apoptosis-regulating genes in RS cells.
Submitted July 1, 1997;
accepted November 13, 1997.
We thank Dr John C. Reed for his generous gift of the anti-Bax polyclonal antibody. We thank Celeste Riley, MD, Winnie Robinson, and Eileen Stein for laboratory help and Maria Ferguson for technical advice. We thank Loesje Troglia for technical help with manuscript preparation and Metin Ozdemirli, MD, for valuable discussions.
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| Copyright © 1998 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Abstract
Introduction
Abstract
B (NF
20°C.
