|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
NEOPLASIA
From the Institute of Pathology, Consultation and
Reference Centre for Lymph Node Pathology and Haematopathology,
University Hospital Benjamin Franklin, Free University, Berlin,
Germany, and the Department of Physiological Chemistry,
Universität Ulm, Ulm, Germany.
The absence of immunoglobulin (Ig) expression in B-cell-derived
Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease
(cHD) was initially suggested to be caused by crippling mutations in
the Ig promoter or coding region. More recent investigations have,
however, challenged this concept. This study addressed the role of
mutations in the Ig promoter region in HRS cells. Nine cases of cHD and
3 B-cell-derived HD lines were analyzed for mutations in the TATA box
and octamer motif of the Ig promoter. Mutations in the octamer motif
were found in only 1 of the 9 cases and in 1 of the 3 HD cell lines
(L1236). Furthermore, in all cases either a complete lack or strong
reduction in the expression of the Oct2 transcription factor and the
BOB.1/OBF.1 coactivator were found. The relevance of the rare promoter
mutations was investigated by assaying the activity of Ig promoter
reporter constructs transfected into the HD cell line L1236, which
harbors a mutated octamer motif. These Ig reporter constructs were
completely inactive in L1236 cells; however, their activity could be
reconstituted by the cotransfection of a BOB.1/OBF.1 expression vector.
The additional transfection with an Oct2 expression vector did not
further enhance the Ig promoter activity. The conclusions drawn from
these results are that crippling mutations in the Ig promoter and
coding region are not the sole cause for the lack of Ig expression in
HRS cells and that defects in the transcription machinery such as
absence of BOB.1/OBF.1 are more important for this phenomenon.
(Blood. 2001;97:3191-3196) It has recently been demonstrated that classical
Hodgkin disease (cHD) is, in approximately 98% of cases, a B-cell
lymphoma,1 and in about 2% of cases a T-cell
lymphoma.2 In light of these findings it is still
unaccountable why B-cell-type Hodgkin and Reed-Sternberg (HRS) cells
do not show any (or show only a very low level of) transcription of the
immunoglobulin (Ig) light (L)1,3,4-6 and heavy
(H) chain genes,1 although they contain
rearranged IgL and IgH genes1,7
There are 4 possible explanations for this phenomenon: (1) crippling
mutations within the V region gene render the coding
sequence nonfunctional,8 (2) crippling mutations in the
octamer motif of the Ig promoter or other regulatory sequences inactivate the Ig transcription,9 (3) defects in Ig
transcription factors prevent Ig gene
transcription,1 or (4) any combination of these. In a
recent study1 we demonstrated that crippling mutations in
the Ig coding region are absent in the majority of cases, making it
unlikely that crippling mutations within the V region gene
are generally responsible for the lack of Ig gene transcription in HRS cells. We also demonstrated that the 2 HD-derived cell lines, KM-H2 and L428, have largely lost their Ig gene
transcription ability, supporting the view that the Ig transcription
machinery might indeed be defective in HRS cells. Support for a role of alterations in the Ig octamer-binding region came from work by Jox and
coworkers9 in which a mutation in the octamer region of
the HD-derived cell line L1236 was reported, raising the possibility that mutations in the Ig gene regulatory element might
contribute to the blockage of Ig transcription in HRS
cells.10,11
To clarify the role of alterations in the Ig regulatory sequences
conclusively, we investigated the TATA box and the octamer motif of the
Ig promoters in single HRS cells isolated from 9 HD cases with
functional (n = 6) and nonfunctional (n = 3) Ig genes
and with absence of Ig expression. Furthermore, in addition to the Ig
promoter region, we studied the intronic Eµ enhancer in the 3 HD cell
lines with rearranged Ig genes. Moreover, these cell lines
were also analyzed for expression of the Ig transcription factors Oct1,
Oct2, and BOB.1/OBF.1 as well as for mutations in the coding regions of
these factors. Finally, the activity of Ig promoter-driven reporter
constructs or synthetic octamer-dependent reporters transfected into
L1236 cells with or without cotransfection of BOB.1/OBF.1 and/or Oct2
was determined. The results obtained from these experiments show that
mutations in the Ig regulatory motifs are rare and do not significantly
contribute to the absence of Ig expression. Instead, the absence of
BOB.1/OBF.1 appears to be the most decisive cause for the lack of Ig
expression in classical HD.
Cell culture and transfection
Single cell isolation
PCR Amplification of the Ig promoter.
The Ig promoter region comprising heptamer and octamer motifs, TATA
box, and pyrimidin region as well as parts of the coding region
(leader, FW1, and CDR1) were amplified from single cells and from
HD-derived cell lines by seminested (PCR; Figure
1). The upper strand primers were located
between 160 to 230 bp (depending on the VH segment involved in the Ig
rearrangement) upstream of the leader1 segment13,14 The
lower strand primers were placed in highly mutated CDR2 and FW3
sections of the already sequenced VH rearrangements.1 This
ensured the nearly exclusive amplification of the promoter of the
rearranged Ig gene. PCR was performed with 40 cycles of amplification
for both rounds of PCR. However, each case required the selection of
individual primers and thus different conditions for primer annealing
and MgCl2 concentration. The initial 5 cycles of the first
PCR consisted of 96°C for 15 seconds, 60°C to 64°C for 150 seconds, and 72°C for 40 seconds. In the remaining 35 cycles, the
cycling conditions were kept constant except for the annealing
temperature (56°-59°C) and time (60 seconds). The reamplification
was carried out with an aliquot (1%) of the first PCR under the same
conditions but with higher annealing temperatures throughout the PCR
(60°-65°C) The buffer conditions were kept constant throughout both
rounds of PCR consisting of 1.5 to 2.5 mM MgCl2 (depending
on the case-specific primers), 150 ng forward primer, 100 ng reverse
primer, and 0.8 mM each dNTP in Taq buffer supplied with 2 U Taq
polymerase (Applied Biosystems, Weiterstadt, Germany).
Amplification of the intronic Eµ enhancer. The amplification of an approximately 500-bp proportion of the intronic Eµ enhancer was carried out with DNA extracted from 3 HD-derived cell lines using a seminested PCR approach. Briefly, the first round of amplification was performed with the upstream primer ENHup 5'-ACA GCA GGT GGC AGG AAG-3' (100 ng) and the downstream primer ENHlow 5'-AAA ATG AAG GCC ACT CTA TCA G-3' (150 ng). In the second round of amplification the ENHup was replaced by the upstream primer ENHRup (5'-CAC CGC GAG AGT CTA TTT TAG G-3'; 100 ng). The cycling conditions consisted of 5 initial cycles at 96°C for 15 seconds, 60°C for 120 seconds, and 72°C for 60 seconds and 35 further cycles with an annealing at 56°C for 60 seconds. The cycling conditions were kept constant in the second round of amplification with the exception that the primer annealing was performed at 60°C throughout the PCR. The buffer conditions were identical throughout both rounds of PCR consisting of 2 U Taq polymerase (Perkin-Elmer), 2.5 mM MgCl2, and 0.8 mM each dNTP. Amplification of transcribed Ig rearrangements. Reverse transcriptase PCR (RT-PCR) for the amplification of the Ig transcripts was performed on HD-derived cell lines as well as on control B-cell and T-cell lines. For this purpose, total RNA (1 µg) was transcribed into single-stranded complementary DNA (cDNA) with random hexamer oligonucleotides and reverse transcriptase. For the first round of amplification a set of 6 family-specific framework (FW) I primers was applied in combination with a JH-specific consensus primer (LJH). Aliquots (6 µL) of the first PCR were analyzed on 6% polyacrylamide gels for the presence of Ig-specific PCR products. For the detection of very small amounts of Ig transcripts, a reamplification was performed with 1 µL of the first PCR as a template using a set of family-specific FWII primers in conjunction with a second JH-specific primer (VLJH). The conditions for the first and second PCR were as described elsewhere.1 The PCR products of Ig promoter, intronic Eµ enhancer, and rearranged Ig genes were separated on 0.8% agarose gels, isolated, and sequenced in both directions (BigDye sequencing system, Applied Biosystems) using the primers of the reamplification separately.RT-PCR for the detection of Oct1, Oct2, and BOB.1/OBF.1 The RT-PCR for the detection of Oct1, Oct2 and BOB.1/OBF.1 transcripts was performed with RNA from various cell lines. In brief, 1 µg total RNA was subjected to 30 cycles (Oct2) or 40 cycles (Oct1 and BOB.1/OBF.1) of PCR after reverse transcription. The primers used for amplification of Oct1, Oct2, and BOB.1/OBF.1 RNA were located within the coding region (Oct1 divided in to 3 parts: 5'-segment-up 5'-CAG CAG CAG CAG ACT CAA G-3', 5'-segment-low 5'-GTC TTG GCA AAC TGC TCA AG-3'; middle segment-up 5'-GCC CAG TGA CCT TGA GGA G-3', middle segment-low 5'-ACG TGA CTG CTG AGG AAG C-3'; 3'-segment-up 5'-CAC AGC AAC CGT GAT TTC C-3', 3'-segment-low 5'-GCT TCT GGC AGC CCA GC-3'; Oct2-up 5'-CCT GCT CAG TTC CTG CTA CC-3' and Oct2-low 5'-GAT GCT GGT CCT CTT CTT GC-3'; BOB.1-up 5'-CAT CCT GTC ACA GGC CAT G-3' and BOB.1-low 5'-AGG ACT CAG GTG GGA GCC AC-3'). The conditions for PCR consisted of 30 seconds at 95°C, 40 seconds at 60°C (Oct1), 63°C (Oct2), or 56°C (BOB.1) and 40 seconds at 72°C. All reactions were performed with 2 U Taq polymerase (Perkin-Elmer) and Taq buffer supplemented with 1.5 mM MgCl2 and 0.8 mM each dNTP and 100 ng of each primer. The PCR products were cloned by using the TOPO TA Cloning Kit (Invitrogen, San Diego, CA) and sequenced in both directions.Northern blot analysis Conditions for Northern blots using 10 µg total RNA and probes specific for BOB.1/OBF.1, Oct1, Oct2, and GAPDH were as described.12Western blot analysis and electromobility shift assays For the detection of Oct2 and BOB.1/OBF.1 proteins in various cell lines, 100 µg whole cell extracts were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel, proteins were transferred to polyvinylidene difluoride (PVDF) membrane and incubated with polyclonal rabbit anti-BOB.1/OBF.1 antibodies,12 Oct1 and Oct2 antibodies (Santa Cruz Technologies, Santa Cruz, CA). After incubation with appropriate secondary antibodies blots were developed using the enhanced chemiluminescence detection system (Amersham, Uppsala, Sweden). The DNA-binding activity of Oct2 was determined as previously described12 using whole cell extracts of L1236, L428, and KM-H2 as well as Raji and Namalwa (controls).
Immunoglobulin transcripts in HD-derived cell lines The expression of Ig genes in HD-derived cell lines was analyzed by RT-PCR. No PCR products were detectable after the first round of amplification in all T-cell-derived as well as in all B-cell-derived HD cell lines, whereas all control B-cell lines investigated were clearly positive. After reamplification, the control B-cell lines gave huge amounts of PCR products in contrast to the B-cell-derived HD cell lines L1236, L428, and KM-H2, which displayed only faint Ig-specific PCR products. All T-cell-derived HD cell lines and T-cell lines were completely negative. This indicates that B-cell-derived HD cell lines contain only traces of Ig messenger RNA (mRNA).Mutations in IgH promoter and coding regions of cultivated and primary Reed-Sternberg cells Cell lines.
To elucidate the reasons for the near complete absence of Ig
transcripts in B-cell-derived HD cell lines, we studied their Ig
promoter region comprising TATA box and octamer motif as well as their
intronic Eµ enhancer site. All HD-derived cell lines were devoid of
mutations with the exception of the already described mutation in the
octamer binding site of the cell line L12369 (Table
1) and a few mutations outside the
transcription factor recognition sites, which most likely represent
irrelevant interindividual polymorphisms (not shown).
Primary HRS cells.
To determine the frequency of mutations in the IgH promoter region in
primary tissues, 335 single HRS cells were isolated by
micromanipulation from immunostained (CD30) frozen sections of 9 cases
of nodular sclerosing HD whose rearranged VH region sequences were
known from a previous study.1 The VH promoter regions
containing the octamer motif and the TATA box were successfully amplified from 65 HRS cells. The amplification of the correct Ig
promoter segment was confirmed by a sequence overlap of the amplified
proportion with the known VH sequences in these cases. Mutations in the
octamer region were found only in 1 of the 9 cases (Table
2), whereas the TATA box was found to be
devoid of mutations in all instances. In addition, single base
substitutions were present outside the transcription factor binding
sites, most likely representing interindividual modifications
(not shown).
VH mutations. The largest proportion of the rearranged IgH coding regions (FW2 to JH) of the 9 classical HD cases used for this study have already been described in previous publications1 showing functional Ig rearrangements in 6 cases and crippling Ig mutations in 3 cases. In the present study, we extended the analysis of the Ig coding region from leader 1 to FW2. No further crippling mutations were found in this proportion of the Ig coding region of all cases, although there was a high load of mutations in most instances (Table 2). Analysis of Ig gene promoter activity in L1236 cells The lack of Ig gene transcription in L1236 cells has been attributed to the mutation in the octamer motif. Therefore, we wondered whether a wild-type Ig promoter would be active in L1236 cells. Reporter constructs driven by either wild-type or mutated Ig promoters were transfected into L1236 cells (Figure 2D). As a control, the B-cell lines expressing Ig on the RNA and protein level were investigated in parallel. Following transfection into the control B-cell lines, the wild-type construct was 12 to 18 times more active than the mutated promoter (Figure 2A). Strikingly, in the L1236 cells the wild-type promoter was as inactive as the mutated one (Figure 2A). To clarify the cause of this promoter inactivity, we transfected synthetic luciferase Ig promoter constructs driven by multimerized octamer motifs. Again, the unmutated promoter construct showed a high activity in the control B-cell lines but virtually no activity in the L1236 cells (Figure 2C). This absence of Ig promoter activity was, however, not due to a general transcription deficiency as shown by transfection of L1236 cells with a herpes simplex virus thymidine kinase promoter. This construct revealed the same level of activity in the L1236 cells as in the control B-cell line (Figure 2B).
Analysis of the transcription factors Oct1 and Oct2 and their coactivator BOB.1/OBF.1 A potential explanation for the lack of Ig promoter activity could be the absence of the Oct transcription factors or BOB.1/OBF.1 or both. We therefore investigated their expression at the RNA (Northern blotting and RT-PCR) and protein level (immunohistochemistry, Western blotting, and electromobility shift assays [EMSAs] and super shift assays).Transcripts specific for Oct2 were completely absent from the
HD-derived cell lines L1236 and L428 and only low levels were detectable in the cell line KM-H2. BOB.1/OBF.1 transcripts were not
present in KM-H2 and L428 and were expressed at very low levels in
L1236 cell line cells. In contrast, the methods applied revealed significant amounts of mRNA for Oct2 and BOB.1/OBF.1 in all 3 sIg+ B-cell lines investigated for control. Oct1 mRNA was
present in all B-cell and HD-derived cell lines (Table
3).
The analysis of the protein expression revealed absence of BOB.1/OBF.1
and Oct2 in all 3 HD-derived cell lines, whereas an expression of these
factors was easily detectable in all control B-cell lines (Figure
3 and Table 3). The absence of Oct2
protein was confirmed by EMSA. The 3 HD-derived cell lines did not
exhibit any Oct2 DNA-binding activity in contrast to the control B-cell lines (Figure 4). Oct1 protein was
present in large amounts in all cell lines (Figure 4).
Cotransfection with BOB.1/OBF.1 and Oct2 To elucidate whether the down-regulation of BOB.1/OBF.1 and Oct2 is causally involved in silencing Ig expression in classical HD, we cotransfected both transcription factors into L1236 cell line cells containing wild-type or mutated Ig promoter constructs. Transfection of BOB.1/OBF.1 but not of Oct2 reestablished the activity of the wild-type promoter. Cotransfection of both BOB.1/OBF.1 and Oct2 did not further enhance the promoter activity. The transfection of BOB.1/OBF.1 or Oct2 (or both) had no effect on the mutated Ig promoter construct demonstrating the specificity of our transfection assay (Figure 5).
We report on studies aimed at clarifying whether mutations in noncoding elements of the IgH gene with special reference to the Ig promoter octamer motif are involved in silencing Ig gene transcription in HRS cells. For this purpose we amplified, by PCR, the TATA box and the octamer motif of the Ig promoter and the Eµ enhancer octamers in 3 bona fide HD cell lines, specifically, L1236, KM-H2, and L428. With the exception of the already described octamer mutation in L1236 cells,9 no mutations were found in the octamer motifs, the TATA boxes, and intronic Eµ enhancers of these cell lines. To determine how generally valid these cell line findings are for classical HD, we extended our investigations to primary HRS cells. For this purpose, we isolated 335 HRS cells by micromanipulation from 9 classical HD cases and analyzed their Ig promoter and neighboring coding sequences. In a previous study we had already analyzed the largest proportion of the Ig coding region (FW2 to JH) of these 9 cases and had found a preserved coding capacity in 6 cases.1 The completion of the analysis of coding regions by amplification with promoter-specific primers in conjunction with HRS cell-specific CDR2 primers revealed no additional crippling mutations in any instance despite the presence of many somatic mutations. The Ig promoter region comprising the TATA box and the octamer motif was unaffected by Ig expression preventing mutations with the exception of one case (case 7) where the octamer motif was modified by 2 base substitutions. This clearly indicates that mutations in the Ig promoter are rare events in primary HRS cells; therefore, they cannot be the general cause for the absence of Ig expression in classical HD. To confirm or dismiss this assumption, we investigated whether a transfected wild-type Ig promoter construct would be active in the L1236 cells containing a mutated endogenous Ig promoter. Strikingly, the transfected wild-type Ig promoter was as inactive in L1236 cells as the mutated promoter transfected for control. In contrast, in control B-cell lines the wild-type promoter showed a 12- to 18-fold higher activity than the mutated promoter. To determine whether this inactivity of the wild-type promoter construct is due to defects in the octamer-dependent transcription, we transfected an Ig promoter construct driven by multimerized octamer motifs. Even with this construct no promoter activity was seen in the L1236 cells, demonstrating that these cells are incapable of activating octamer-dependent transcription. These results, in conjunction with previous data, clearly demonstrate that a defect in the Ig transcription machinery appears to be the real reason for the lack of Ig expression in HRS cells and not mutations in regulatory or coding Ig sequences Because the transcription factors Oct1 and Oct2 and their coactivator BOB.1/OBF.1 are critical for octamer-dependent transcription in B cells,15-17 the question arose whether these factors are missing in classical HD. To test this possibility we investigated the expression of Oct1, Oct2, and BOB.1/OBF.1 at the RNA and protein level in all 3 bona fide HD-derived cell lines (ie, L1236, L428, and KM-H2). Transcripts of Oct2 and BOB.1/OBF.1 proved to be absent (Northern blot analysis) or present at very low levels (RT-PCR). At the protein level neither BOB.1/OBF.1 nor Oct2 was detectable by all methods applied. In contrast, Oct1 was expressed at the RNA and protein level in all HD and control B-cell lines. These findings prompt the conclusion that the missing expression of Oct2 and/or BOB.1/OBF.1 is involved in the down-regulation of Ig expression in classical HD. To further strengthen the validity of this conclusion we cotransfected BOB.1/OBF.1 or Oct2 together with wild-type or mutated Ig reporter constructs into L1236 cells. The transfection of BOB.1/OBF.1 alone completely reconstituted the activity of the wild-type Ig promoter, whereas the transfection of Oct2 had no effect. These findings are in keeping with the observation that Oct1 can compensate the absence of Oct2.18 This conclusion indicates that the lack of BOB.1/OBF.1 is responsible for the missing Ig gene transcription in HRS cells. The importance of BOB.1/OBF.1 was also demonstrated by the observation that both synthetic octamer-dependent promoters as well as a naturally occurring promoter are inactive in B cells that lack the coactivator BOB.1/OBF.1. This inactivity of the Ig promoter can be overcome by induction of BOB.1/OBF.1 expression, demonstrating that BOB.1/OBF.1 is a nonredundant protein in B cells, which is unconditionally required for octamer-dependent transcriptional activity.12 To further strengthen the validity of this conclusion, we
cotransfected BOB.1/OBF.1 or Oct2 together with wild-type or
mutated Ig reporter constructs into L1236 cells. The transfection of
BOB.1/OBF.1 alone completely reconstituted the activity of the
wild-type Ig promoter, whereas the transfection of Oct2 had no or no
additional effect. These findings are in keeping with the observation
that Oct1 can compensate for the absence of Oct2 but appears to be in
conflict with the phenotype of BOB.1/OBF.1-deficient mice because they
show an almost normal development of B cells with expression of normal
levels of IgM.19 However, later stages of B-cell
differentiation are affected by the BOB.1/OBF.1 deficiency causing an
absence of germinal center formation and a 10-fold decrease in serum
isotype levels. Because this reduction does not appear to be due to a defect in class switch recombination,19-21 it might
reflect reduction in the transcription rates at the IgH locus. That
this really is the case could recently be verified by the introduction
of octamer-dependent promoter constructs into BOB.1/OBF.1-deficient cells. These constructs were In summary, we have clearly demonstrated that absent Ig expression is not associated with the presence of crippling mutations in the coding region or Ig promoter. Instead, our data indicate that the coactivator BOB.1/OBF.1 is alone or predominantly responsible for the inactivity of the Ig promoter in HRS cells of cHD.
We thank H. Protz, A. Foerster, and H. Lammert for their excellent technical assistance and L. Udvarhelyi for his help with the preparation of the manuscript.
Submitted October 23, 2000; accepted December 21, 2000.
Supported by grants from the Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe.
J.T. and H.L. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Harald Stein, Institute of Pathology, Benjamin Franklin University Hospital, Free University Berlin, Hindenburgdamm 30, 12200 Berlin, Germany; e-mail: stein{at}ukbf.fu-berlin.de.
1.
Marafioti T, Hummel M, Foss HD, et al.
Hodgkin and Reed-Sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription.
Blood.
2000;95:1443-1450
2.
Seitz V, Hummel M, Marafioti T, Anagnostopoulos I, Assaf C, Stein H.
Detection of clonal T-cell receptor gamma-chain gene rearrangements in Reed-Sternberg cells of classic Hodgkin disease.
Blood.
2000;95:3020-3024 3. von Wasielewski R, Wilkens L, Nolte M, Werner M, Georgii A. Light-chain mRNA in lymphocyte-predominant and mixed-cellularity Hodgkin's disease. Mod Pathol. 1996;9:334-338[Medline] [Order article via Infotrieve]. 4. Ruprai AK, Pringle JH, Angel CA, Kind CN, Lauder I. Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization. J Pathol. 1991;164:37-40[CrossRef][Medline] [Order article via Infotrieve]. 5. Hell K, Lorenzen J, Hansmann ML, Fellbaum C, Busch R, Fischer R. Expression of the proliferating cell nuclear antigen in the different types of Hodgkin's disease. Am J Clin Pathol. 1993;99:598-603[Medline] [Order article via Infotrieve]. 6. Hell K, Pringle JH, Hansmann ML, et al. Demonstration of light chain mRNA in Hodgkin's disease. J Pathol. 1993;171:137-143[CrossRef][Medline] [Order article via Infotrieve].
7.
Kanzler H, Kuppers R, Hansmann ML, Rajewsky K.
Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.
J Exp Med.
1996;184:1495-1505
8.
Kuppers R, Klein U, Hansmann ML, Rajewsky K.
Cellular origin of human B-cell lymphomas.
N Engl J Med.
1999;341:1520-1529
9.
Jox A, Zander T, Kuppers R, et al.
Somatic mutations within the untranslated regions of rearranged Ig genes in a case of classical Hodgkin's disease as a potential cause for the absence of Ig in the lymphoma cells.
Blood.
1999;93:3964-3972 10. Matthias P. Lymphoid-specific transcription mediated by the conserved octamer site: who is doing what? Semin Immunol. 1998;10:155-163[CrossRef][Medline] [Order article via Infotrieve].
11.
Jenuwein T, Grosschedl R.
Complex pattern of immunoglobulin mu gene expression in normal and transgenic mice: nonoverlapping regulatory sequences govern distinct tissue specificities.
Genes Dev.
1991;5:932-943 12. Laumen H, Nielsen PJ, Wirth T. The BOB.1/OBF.1 co-activator is essential for octamer-dependent transcription in B cells. Eur J Immunol. 2000;30:458-469[CrossRef][Medline] [Order article via Infotrieve].
13.
Haino M, Hayashida H, Miyata T, et al.
Comparison and evolution of human immunoglobulin VH segments located in the 3' 0.8-megabase region: evidence for unidirectional transfer of segmental gene sequences.
J Biol Chem.
1994;269:2619-2626
14.
Matsuda F, Ishii K, Bourvagnet P, et al.
The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus [see comments].
J Exp Med.
1998;188:2151-2162
15.
Staudt LM, Clerc RG, Singh H, LeBowitz JH, Sharp PA, Baltimore D.
Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif.
Science.
1988;241:577-580 16. Luo Y, Fujii H, Gerster T, Roeder RG. A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors. Cell. 1992;71:231-241[CrossRef][Medline] [Order article via Infotrieve]. 17. Luo Y, Roeder RG. Cloning, functional characterization, and mechanism of action of the B-cell-specific transcriptional coactivator OCA-B. Mol Cell Biol. 1995;15:4115-4124[Abstract]. 18. Shah PC, Bertolino E, Singh H. Using altered specificity Oct-1 and Oct-2 mutants to analyze the regulation of immunoglobulin gene transcription. EMBO J. 1997;16:7105-7117[CrossRef][Medline] [Order article via Infotrieve]. 19. Schubart DB, Rolink A, Kosco-Vilbois MH, Botteri F, Matthias P. B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation. Nature. 1996;383:538-542[CrossRef][Medline] [Order article via Infotrieve]. 20. Kim U, Qin XF, Gong S, et al. The B-cell-specific transcription coactivator OCA-B/OBF-1/Bob-1 is essential for normal production of immunoglobulin isotypes. Nature. 1996;383:542-547[CrossRef][Medline] [Order article via Infotrieve]. 21. Nielsen PJ, Georgiev O, Lorenz B, Schaffner W. B lymphocytes are impaired in mice lacking the transcriptional co-activator Bob1/OCA-B/OBF1. Eur J Immunol. 1996;26:3214-3218[Medline] [Order article via Infotrieve]. 22. Pfisterer P, Annweiler A, Ullmer C, Corcoran LM, Wirth T. Differential transactivation potential of Oct1 and Oct2 is determined by additional B cell-specific activities. EMBO J. 1994;13:1655-1663[Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  D. L. Di Bartolo, E. Hyjek, S. Keller, I. Guasparri, H. Deng, R. Sun, A. Chadburn, D. M. Knowles, and E. Cesarman Role of Defective Oct-2 and OCA-B Expression in Immunoglobulin Production and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation in Primary Effusion Lymphoma J. Virol., May 1, 2009; 83(9): 4308 - 4315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G Kapatai and P Murray Contribution of the Epstein Barr virus to the molecular pathogenesis of Hodgkin lymphoma J. Clin. Pathol., December 1, 2007; 60(12): 1342 - 1349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ushmorov, F. Leithauser, O. Sakk, A. Weinhausel, S. W. Popov, P. Moller, and T. Wirth Epigenetic processes play a major role in B-cell-specific gene silencing in classical Hodgkin lymphoma Blood, March 15, 2006; 107(6): 2493 - 2500. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Atayar, S. Poppema, T. Blokzijl, G. Harms, M. Boot, and A. van den Berg Expression of the T-Cell Transcription Factors, GATA-3 and T-bet, in the Neoplastic Cells of Hodgkin Lymphomas Am. J. Pathol., January 1, 2005; 166(1): 127 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ushmorov, O. Ritz, M. Hummel, F. Leithauser, P. Moller, H. Stein, and T. Wirth Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression Blood, November 15, 2004; 104(10): 3326 - 3334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Marafioti, M. Pozzobon, M.-L. Hansmann, G. Delsol, S. A. Pileri, and D. Y. Mason Expression of intracellular signaling molecules in classical and lymphocyte predominance Hodgkin disease Blood, January 1, 2004; 103(1): 188 - 193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Re, P. Starostik, N. Massoudi, A. Staratschek-Jox, V. Dries, R. K. Thomas, V. Diehl, and J. Wolf Allelic Losses on Chromosome 6q25 in Hodgkin and Reed Sternberg Cells Cancer Res., May 15, 2003; 63(10): 2606 - 2609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Marafioti, S. Ascani, K. Pulford, E. Sabattini, M. Piccioli, M. Jones, P. L. Zinzani, G. Delsol, D. Y. Mason, and S. A. Pileri Expression of B-Lymphocyte-Associated Transcription Factors in Human T-Cell Neoplasms Am. J. Pathol., March 1, 2003; 162(3): 861 - 871. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Diehl, H. Stein, M. Hummel, R. Zollinger, and J. M. Connors Hodgkin's Lymphoma: Biology and Treatment Strategies for Primary, Refractory, and Relapsed Disease Hematology, January 1, 2003; 2003(1): 225 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. F. Dukers, C. J. L. M. Meijer, R. L. ten Berge, W. Vos, G. J. Ossenkoppele, and J. J. Oudejans High numbers of active caspase 3-positive Reed-Sternberg cells in pretreatment biopsy specimens of patients with Hodgkin disease predict favorable clinical outcome Blood, June 17, 2002; 100(1): 36 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Jundt, K. Kley, I. Anagnostopoulos, K. Schulze Probsting, A. Greiner, S. Mathas, C. Scheidereit, T. Wirth, H. Stein, and B. Dorken Loss of PU.1 expression is associated with defective immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease Blood, April 15, 2002; 99(8): 3060 - 3062. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-mercaptoethanol at 37°C and
5% CO2. For electroporation 5 × 106 cells
were pelleted and resuspended in a final volume of 300 µL RPMI medium
supplemented with 10% FCS. Fifteen micrograms of the respective
luciferase reporter plasmid
109
to +52) or a truncated version of this promoter (pTATA, 
like in HRS cells