Haematologica 2003 - Supplements

detected in 293 and NIH3T3 cells. Similarly, MMSET transgenic
protein was detected in T cells from thymus, lymph nodes and
spleen of the transgenic mice, but not in any B cells, whose
number and phenotype were otherwise normal, excluding an
MMSET inhibitory effect on B cell development. We conclude
that MMSET protein expression must be tightly regulated in B
cells and that it shares more features in common with a tumor
suppressor gene than with an oncogene.
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Cancer-Related Potential Target Genes of MMSET in
Multiple Myeloma
Heidi Rye Hudlebusch1, Kim Theilgaard-Mönch2, Marianne
Lohdahl1, Hans Erik Johnsen1, Thomas Rasmussen1.
1) The Department of Hematology, Herlev University Hospital,
Denmark; 2) The Granulocyte Res. Lab., Rigshospitalet, Denmark
In Multiple Myeloma (MM) the frequently detected translocation
t(4;14)(p16.3;q32) results in a dysregulation of two potential
oncogenes, the recently discovered MM SET domain (MMSET)
and the fibroblast growth factor receptor 3 (FGFR3). MMSET is
a putative transcription factor expressed as two mRNA isoforms,
a short form (type I) and a long form (type II). MMSET is a
member of the trithorax group of nuclear proteins involved in
modulation of chromatin remodelling based on the presence of
the SET domain and of four plant homeo domian (PHD) zinc
fingers. In this study the oncogenic role of MMSET in MM has
been examined by defining potential target genes of MMSET by
a cDNA array analysis.
To investigate the role of MMSET in MM, MMSET type I and II
were cloned into a pCMS-EGFP mammalian expression vector.
The vectors containing MMSET type I or II were transfected into
the human erythroleukemia cell line, K562, expressing MMSET
at a low level. Successful transfection was documented by flow
cytometry detection of the enhanced green fluorescent protein
(EGFP) and subsequent MMSET quantitation by real-time RT-
PCR. The EGFP+ K562 cells transfected with the vectors
containing MMSET type I or II and with the empty vector were
FACS sorted after 6, 12 and 24 h and RNA was extracted. After
verifying a high level of MMSET type I or II expression, gene
expression profiling was performed by cDNA arrays (U133A,
Affymetrix). So far, we have identified 25 genes with a
significant upregulation caused by MMSET type I. 3/25 genes
(CLI/Clusterin, FOSB and JUN) were selected for further
analysis because of their described association with cancer. To
examine whether these genes had a relation to MMSET
dysregulation in primary MM tumors a quantitative detection by
real-time RT-PCR was performed on FACS purified plasma cells
from MM patients with an upregulation of either MMSET (n=4)
or cyclin D1 (n=4) as detected by real-time RT-PCR. The
presence of the t(4;14) translocation in the MMSET+ patients
was verified by detection of the IgH-MMSET hybrid transcript.
The results documented that an upregulation of CLI/Clusterin,
FOSB and JUN was detected exclusively in the patient samples
with a MMSET dysregulation. The downstream upregulation of
cancer-related genes supports the hypothesis that MMSET is an
oncogene.
004
Secondary Translocations Dysregulate c-, N-, or L-MYC
in Multiple Myeloma
Amel Dib, Ying Qi, Leslie Brents, Yaping Shou, Marina
Martelli, Ana Gabrea, P. Leif Bergsagel, and Michael Kuehl
Genetics Branch, National Cancer Institute
Simple, reciprocal chromosomal translocations juxtaposing c-
MYC to an Ig locus are an invariant event in murine
plasmacytoma tumors. These primary Ig translocations occur
early in tumorigenesis as a result of errors in one of two B cell
specific DNA modification processes (IgH switch recombination
or somatic hypermutation). Expression of the translocated c-
MYC allele is dysregulated, whereas the normal c-MYC allele is
silent, corresponding to the absence of c-MYC expression in
terminally differentiated plasma cells. Although primary Ig
translocations are found in a majority of multiple myeloma (MM)
tumors, these translocations rarely – if ever - involve c-MYC.
Instead, dysregulation of c-, N-, or L-MYC is mediated by
secondary (Ig) translocations as a late progression event in MM.
Cloned translocation breakpoints infrequently occur within or
near V(D)J or switch sequences, consistent with a lack of
involvement of B cell specific DNA modification processes.
Three color FISH analyses of metaphase chromosomes showed
complex translocations and insertions of c-MYC, L-MYC (one
cell line), or N-MYC (one tumor) in 25 of 29 (86%) human MM
cell lines (HMCL) and 18 of 38 (47%) MM tumors examined.
These karyotypic abnormalities often are non-reciprocal and
frequently involve 3 chromosomes, sometimes with associated
inversions, deletions, or duplications. Surprisingly, only half of
the karyotypically abnormal MYC loci include associated Ig
sequences. The MYC karyotypic abnormalities are associated
with dysregulation of a single allele, since all 13 informative MM
cell lines express either L-MYC or one of two genetically
distinguishable c-MYC alleles; and 2/82 primary tumors express
N-MYC but not c- or L-MYC. Corroborative studies by Avet-
Loiseau et al have shown that similar karyotypic abnormalities of
c-MYC are detected by interphase FISH in 3% of MGUS tumors
and 15% of MM tumors, often with heterogeneity within a single
tumor specimen. Together, these studies indicate that the
dysregulation of one MYC allele is mediated by a complex
translocation that is associated with increased proliferation and
autonomy from the influence of bone marrow stromal cells. We
have used a combination of FISH mapping, and molecular
cloning to better understand the structures of complex
translocations that do or do not involve an Ig enhancer.
Translocations can have breakpoints that occur up to 1 Mb
telomeric or centromeric to c-MYC. Also, as little as 100 kb from
the c-MYC locus can be inserted at other chromosomal locations,
including translocation breakpoints that involve two other
chromosomes. For correlations of structure and expression, we
have also used a combination of DNA and RNA FISH on HMCL,
and also constructed HMCL X mouse plasmacytoma hybrids to
correlate abnormal chromosome structures with human MYC
expression. A summary of our results will be presented, including
the Karpas 620 HMCL that apparently underwent sequential
t(11;14) and t(8;11;14) translocations involving the same IgH
locus. Large regions of chromosomes 11 and 8 were duplicated
during the second translocation: der14 t(14;11;8) contains and
expresses c-MYC; whereas der(8) t(8;14;11) contains c-MYC
and CYCLIN D1 but expresses only CYCLIN D1 and not c-
MYC. The implications of these various results will be discussed.
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