Martin Janz ^{1, 2,} *, Michael Hummel ³, Matthias Truss ⁴, Brigitte Wollert-Wulf ^{1, 2}, Stephan Mathas ^{1, 2}, Korinna Johrens ³, Christian Hagemeier ⁴, Kurt Bommert ^{1, 2}, Harald Stein ³, Bernd Dorken ^{1, 2}, and Ralf C Bargou ^{1, 2}

¹ Max Delbruck Center for Molecular Medicine, Berlin, Germany;
² Department of Hematology, Oncology, and Tumorimmunology, Charite, University Medicine Berlin, Campus Buch and Campus Virchow-Klinikum, Berlin, Germany
³ Institute of Pathology, Charite, University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany
⁴ Department of Pediatrics, Laboratory for Molecular Biology, Charite, University Medicine Berlin, Berlin, Germany

* Address correspondence to:
Martin Janz, M.D., Hematology, Oncology, and Tumorimmunology, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13092 Berlin, Germany, Tel./Fax: +49-30-9406-3244
email: mjanz@mdc-berlin.de

Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) display unique characteristics that discriminate cHL from other B cell lymphomas and normal B cells. Therefore, comparative gene expression profiling of Hodgkin and non-Hodgkin B cells could lead to the identification of candidate genes that are critical for the pathogenesis of cHL. We performed microarray analysis of Hodgkin and non-Hodgkin cell lines and identified the transcription factor ATF3, a member of the CREB/ATF family, as a differentially expressed candidate gene. Extensive analysis of a large panel of cell lines, primary tumor samples, and normal tissues revealed that high expression of ATF3 is found in nearly all cases of cHL and is almost exclusively restricted to it. Selective knock-down of ATF3 by RNA interference suppressed proliferation and strongly reduced viability of Hodgkin cells. Thus, overexpression of ATF3 is a molecular hallmark of cHL that contributes to the malignant growth of HRS cells.

Classical Hodgkin lymphoma (cHL) is a common malignant lymphoma which is
characterized by the presence of typical malignant cells, the mononuclear Hodgkin and
multinuclear Reed-Sternberg (HRS) cells [1]. HRS cells, which represent only a minor cellular
fraction of the affected lymphoid tissue, are thought to be derived from germinal or post-germinal
center B cells [2, 3]. Although the precise molecular mechanisms underlying the
malignant transformation of HRS cells are only partially understood, several observations
indicate that defects in transcriptional regulation play a pivotal role in the pathogenesis of
cHL. In line with this idea, we have previously identified high constitutive activity of the
transcription factor NF-kB as a common characteristic of HRS cells that contributes to their
survival and proliferation [4, 5]. In non-malignant cells, NF-kB is only transiently activated,
particularly by stress signals and in immune and inflammatory signaling events [6]. Another
hallmark of cHL is the aberrant expression of the AP-1 transcription factor family members c-Jun
and JunB [7] which are usually activated in response to mitogenic stimuli and cellular stress
signals.

HRS cells display unique morphological and molecular characteristics that clearly
discriminate cHL from B cell non-Hodgkin lymphomas and normal B cells [8]. Therefore, gene
expression profiling is a promising approach to identify genes that contribute to malignant
growth and survival in cHL. We performed oligonucleotide microarray analysis of Hodgkin
and non-Hodgkin cell lines and identified activating transcription factor 3 (ATF3) as a
differentially expressed candidate gene with high expression in HRS cells. ATF3 is a member
of the ATF/CREB family of basic region leucine zipper transcription factors which is
involved in the cellular stress response [9]. ATF3 expression is strongly induced by cellular
damage in a variety of organ systems, including liver, heart, and neurons as well as in cultered
cells following exposure to UV light, ionizing radiation, proteasome inhibitors, or interference
with protein synthesis. Furthermore, ATF3 has been reported to be associated with cellular
proliferation and survival [10,11,12]. We therefore decided to analyze the role of ATF3 in cHL in
more detail.

Study design:

Cell lines, electroporation, and purification of transfected cells

Cell lines, culture conditions, and electroporation have been described previously [7]. Where
indicated, an additional purification step following transfection was included, employing
either fluorescence-activated cell sorting (FACS) or magnetic cell sorting (MACS) as detailed
in Mathas et al [7] and Chatterjee et al [13].

Oligonucleotide microarray analysis

RNA processing and hybridization to U95A GeneChip microarrays was performed according
to the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Primary data files were
analyzed with the Microarray Suite software (Affymetrix). Hierarchical clustering analysis
was carried out with the Cluster and TreeView software (M. Eisen, Berkeley, CA) [14]. .

Northern and Western blot analysis

Northern and Western blotting was performed according to standard protocols as described
previously [7]. The following primary antibodies were used: anti-ATF3 (sc-188; Santa Cruz,
CA), anti-E2F-4 (clone 4E2F04; Neomarkers, CA), and anti-a -tubulin (MCA78A; Serotec,
München, Germany).

Immunohistochemistry

All cases were retrieved from the files of the Institute of Pathology, Charité, Campus
Benjamin Franklin, Berlin, Germany. Diagnosis was established after histological and
immunohistological analysis according to the criteria of the World Health Organization
classification. ATF3 immunohistochemistry was performed with a polyclonal rabbit anti-ATF3
antibody (sc-188; Santa Cruz, CA) after heat pretreatment of the deparaffinized tissue
sections in a pressure cooker for 2 minutes in citrate buffer (10 mM). Subsequently, a mouse-anti-
rabbit antibody (DakoCytomation, Glostrup, Denmark) was applied followed by
development employing alkaline phosphatase and fast red as substrate. Approximately two-third
of the tissue sections were arranged in tissue microarrays, whereas the remaining cases
have been stained as single tissue sections.

Construction of siRNA expression plasmids

Vectors for transient expression of small interfering RNAs were derived from the pSUPER
plasmid according to the guidelines described in Brummelkamp et al [15]. The episomal plasmid
vector pRep-H1 (M. Truss et al., manuscript in preparation) was constructed by insertion of a
H1-promoter based PolIII transcription unit into the vector pTIP.HA1x3.EGFP that contains a
puromycin selectable marker, a truncated EBNA gene, and an EBV oriP sequence. The
following target sequences were chosen:
EGFP 5‘-GCAGCACGACTTCTTCAAG-3‘,
Luc (directed against firefly luciferase; pGL2 vectors, Promega)
5‘-CGTACGCGGAATACTTCGA-3‘,
E2F-4 5‘-GCGGCGGATTTACGACATT-3‘,
ATF3_1 5‘-GAGCTGAGGTTTGCCATCC-3‘,
ATF3_2 5‘-GAGGCGACGAGAAAGAAAT-3‘, and
ATF3_3 5‘-GAAGAAGGAGAAGACGGAG-3‘.

Proliferation assay and analysis of apoptosis

DNA synthesis was determined by [ ³H]-thymidine incorporation assays as described in
Lentzsch et al [16]. To assess the percentage of viable and apoptotic cells, annexin V-fluorescein
isothiocyanate (FITC)/propidium iodide (PI) double-staining was performed using a human
annexin V-FITC kit (Bender MedSystems, Eching, Germany). Cells were processed as
described in the manufacturer’s protocol and analyzed by flow cytometry.

Results and discussion:

To identify candidate genes that are involved in the pathogenesis of cHL, we performed
oligonucleotide microarray analysis of Hodgkin and non-Hodgkin B cell lines. Hierarchical
clustering separated Hodgkin from non-Hodgkin cell lines of distinct B cell differentiation
stages. One of the most pronounced up-regulated genes in Hodgkin cell lines was the
transcription factor ATF3 (Suppl. Figure 1). To validate the microarray data on ATF3
expression, Northern and Western blot analysis was carried out on a broad panel of cell lines.
All Hodgkin cell lines demonstrated strong overexpression of ATF3 mRNA and protein
compared to non-Hodgkin cell lines (Figure 1A). ATF3 expression in primary malignant and
non-malignant lymphoid tissue was determined by immunohistochemistry. A total of 415
lymphoma samples were immunostained for ATF3, comprising 71 cases of cHL and 26 cases
of lymphocyte-predominant HL (LPHL) as well as 239 B cell non-Hodgkin lymphomas, 46 T
cell non-Hodgkin lymphomas, and 33 anaplastic large cell lymphomas (ALCL). In addition,
20 specimen of reactive lymphoid tissue were included (Figure 1B, C). Strong nuclear
expression of ATF3 was detected in nearly all cases of cHL analyzed (69/71). Nuclear ATF3
staining was restricted to HRS cells and could not be detected in surrounding bystander cells
(Figure 1B). In contrast, nuclear ATF3 immunoreactivity was observed only in a few samples
of B or T cell non-Hodgkin lymphomas (6/239 and 2/46, respectively), including diffuse large
B cell lymphoma (2/80), plasmacytoma (4/20), and angioimmunoblastic T cell lymphoma
(2/10) (Figure 1C). In these cases, positive staining was found only in a varying, usually small
proportion of the tumor cell population with generally weaker signal intensity compared to
cHL and ALCL. In addition, no nuclear staining was observed in reactive lymphoid tissue
(Figure 1B). Interestingly, ATF3 expression could not be demonstrated in the nuclei of LPHL
tumor cells (Figure 1B). This is in agreement with the notion that cHL and LPHL represent
independent entities which differ in their biology and clinical course. On the other hand,
besides cHL a proportion of ALCL cases demonstrated ATF3 staining (24/33; Figure 1B)
which did not correlate with the expression of ALK fusion proteins (data not shown). ATF3
expression in cHL and ALCL is in line with the observation that these lymphomas share
several features, including CD30 expression as well as high Notch-1 and AP-1 activity [17,18, 7]. It
remains to be determined whether this pattern reflects common pathogenetic pathways in both
lymphoma types. Taken together, our immunohistochemical analysis demonstrated that
nuclear expression of ATF3 is almost exclusively restricted to HRS cells of cHL and to a
fraction of ALCL.

To assess the functional significance of ATF3 overexpression in HRS cells, we selectively
blocked ATF3 expression by RNA interference. Three independent ATF3 siRNA sequences
were chosen that resulted in a significant down-regulation of ATF3 protein level after
transfection into L428 and L540Cy Hodgkin cells (Figure 2A). Several control siRNA
sequences for irrelevant targets had no effect on ATF3 expression (Figure 2A). Next, we
asked whether siRNA-mediated knock-down of ATF3 in Hodgkin cells would affect
proliferation and tumor cell viability. Therefore, we transiently transfected Hodgkin cells with
ATF3-directed siRNA expression plasmids and performed [ ³H]-thymidine incorporation
assays. Down-regulation of ATF3 resulted in a marked reduction of DNA synthesis compared
to control-transfected cells (Figure 2B). To investigate the effects of ATF3 down-regulation
over an extended period of time, we used vector-based siRNA constructs that are propagated
by extrachromosomal replication and carry a puromycin resistance gene permitting the
selection of siRNA-expressing cells. Transfection of L428 and L540Cy Hodgkin cells with
ATF3-directed siRNA plasmids and subsequent antibiotic selection resulted in a significant
and sustained loss of viable cells, as determined by annexin V-FITC/PI double staining and
flow cytometry (Figure 2C). This effect could be induced with different ATF3-targeting
constructs, but not with control siRNA plasmids directed against irrelevant targets. Our
observations on the role of ATF3 in HRS cells are in good accordance with previous reports
that describe a function of ATF3 in the control of cell cycle progression and apoptosis
resistance in other cellular systems. Ectopic expression of ATF3 in hepatocytes stimulates
cyclin D1 expression and proliferation [10]. In addition, ATF3 has been shown to have anti-apoptotic
functions in cardiomyocytes and neuronal cells [19, 12]. In line with its cell cycle-promoting
and anti-apoptotic properties, ATF3 has been shown to partially transform chicken
embryo fibroblasts, indicating an intrinsic oncogenic potential [20]. Thus, in conjunction with
previously published observations on its role in proliferation and survival, our data suggest an
important oncogenic role of constitutive ATF3 expression in cHL.

Acknowledgments:

We are indebted to Reuven Agami (Amsterdam) for the gift of the pSUPER siRNA expression plasmid.

Figure 1: Hodgkin cell lines and primary HRS tumor cells are characterized by high constitutive ATF3 expression.

(A) ATF3 mRNA and protein expression in Hodgkin and non-Hodgkin cell lines was determined by Northern (NB) and Western (WB) blot analysis, respectively. As loading controls, GAPDH mRNA and a -tubulin protein expression were included.

(B) Immunostaining for ATF3 in primary tissue samples (cHL, classical Hodgkin lymphoma; LPHL, lymphocyte-predominant Hodgkin lymphoma; ALCL, anaplastic large cell lymphoma). 

(C) Immunostaining for ATF3 in primary tissue samples

The group of B-cell non-Hodgkin lymphomas (B-NHL) includes 21 mantle cell lymphomas (MCL), 27 B-cell chronic lymphocytic leukemias (B-CLL), 72 follicular lymphomas (FL), 80 diffuse large B-cell lymphomas (DLBCL, including 6 primary mediastinal B cell lymphomas), 19 Burkitt lymphomas (BL), and 20 plasmacytomas. T-cell non-Hodgkin lymphomas (T-NHL) comprise 20 peripheral T-NHLs (PTCL), 10 angioimmunoblastic T-NHLs (AILT), 5 enteropathy-associated T-NHLs, 4 NK/T-cell lymphomas, and 7 cases of Mycosis fungoides.

(A) L428 and L540Cy Hodgkin cells were transfected with control and ATF3-directed pSUPER siRNA expression plasmids, purified and harvested 48 h after electroporation. Whole cell lysates were subjected to Western blot analysis using the indicated antibodies.

(B) L540Cy HRS cells were transfected with the indicated pSUPER siRNA constructs, purified and pulsed with [ 3 H]-thymidine for 24 h before harvesting and assessing incorporated radioactivity. Data are presented as cpm of [ 3 H]-thymidine incorporation (mean +/- SD of five measurements). 

(C) L428 and L540Cy HRS cells were transfected with control and ATF3-directed pRepH1 siRNA expression constructs and selected for siRNA-expressing cells by addition of puromycin 24 h after electroporation. The
percentage of viable and apoptotic cells was determined by annexin V-FITC/propidium iodide (PI) staining and flow cytometry. Viable L428 (left panel) and L540Cy (right panel) cells after 14 days in culture. The fraction of viable cells, negative for both annexin V-FITC and PI, is shown as percentage of total cells. Measurements were performed in triplicate. Error bars indicate SDs.

Gene expression profiles of the indicated cell lines were generated using Affymetrix U95A oligonucleotide microarrays. Hierarchical clustering analysis was performed with the Cluster and TreeView software.

(A) HRS cell lines (KM-H2, L428, L1236, L591) were separated from non-Hodgkin B cell lines in one main branch of the dendrogram. Within the group of non-Hodgkin cell lines, the plasma cell lines (U266, INA-6, MM.1S, L363) were divided from the pre-B cell line Reh and the Burkitt lymphoma cell lines Namalwa and BL60.

http://www.bloodjournal.org/cgi/content/full/2005-07-2694/DC1

(B) Expanded view of the cluster that most clearly separates between HRS and non-Hodgkin cells. Relative ATF3 expression across Hodgkin and non-Hodgkin cell lines is indicated (*).

This new and comprehensive report by Martin Janz, Michael Hummel, Matthias Truss, Brigitte Wollert-Wulf, Stephan Mathas, Korinna Johrens, Christian Hagemeier, Kurt Bommert, Harald Stein, Bernd Dorken, and Ralf C Bargou, reveals the overexpression of ATF3 in classical Hodgkin lymphoma neoplastic cells, but not in other lymphoma neoplastic cells, nor in other normal lymphatic tissues. ATF3 is a known oncogene in other systems, and its overexpression may play a similar oncogenic role in Hodgkin lymphoma. Specific anti-ATF3 siRNA knockdown of ATF3 in Hodgkin neoplastic cells results in decreased DNA synthesis and decreased viability of Hodgkin lymphoma neoplastic cells. Anti-Oncogenes may be effective agents against Oncogenes within human neoplastic cells.

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"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).