Relevance of cyclin D1b expression and CCND1polymorphism in the pathogenesis of multiple myeloma and mantle cell lymphoma
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The CCND1 gene generates two mRNAs (cyclin D1a and D1b) through an alternative splicing at the site of a common A/G polymorphism. Cyclin D1a and b proteins differ in their C-terminus, a region involved in protein degradation and sub-cellular localization. Recent data have suggested that cyclin D1b could be a nuclear oncogene. The presence of cyclin D1b mRNA and protein has been studied in two hemopathies in which cyclin D1 could be present: multiple myeloma (MM) and mantle cell lymphoma (MCL). The A/G polymorphism of CCND1 has also been verified in a series of patients.
The expression of cyclin D1 mRNA isoforms has been studied by real-time quantitative PCR; protein isoforms expression, localization and degradation by western blotting. The CCND1 polymorphism was analyzed after sequencing genomic DNA.
Cyclin D1 mRNA isoforms a and b were expressed in mantle cell lymphoma (MCL) and multiple myeloma (MM). Cyclin D1b proteins were present in MCL, rarely in MM. Importantly, both protein isoforms localized the nuclear and cytoplasmic compartments. They displayed the same short half-life. Thus, the two properties of cyclin D1b recognized as necessary for its transforming activity are missing in MCL. Moreover, CCND1 polymorphism at the exon/intron boundary had no influence on splicing regulation in MCL cells.
Our results support the notion that cyclin D1b is not crucial for the pathogenesis of MCL and MM.
KeywordsMultiple Myeloma Mantle Cell Lymphoma Mantle Cell Lymphoma Cell CCND1 Gene Mantle Cell Lymphoma Patient
Cyclin D1 is a critical regulator of the cell cycle and transcriptional processes. Overexpression or overactivity of cyclin D1 is common in human cancers. Cyclin D1 is also expressed in lymphoid tumors such as mantle cell lymphoma (MCL) and multiple myeloma (MM). The expression of cyclin D1 in B-cells in which the protein is physiologically absent, is supposed to be causal in the transformation process. In MCL, CCND1 (encoding cyclin D1) is activated by the t(11;14)(q13;q32) in almost 100% of cases . In multiple myeloma (MM), cyclin D1 is expressed in 45–50% of samples but the t(11;14)(q13;q32) is only present in 15% of them . The human CCND1 gene encodes two mRNAs species resulting from an alternative splicing: form a  and form b generated by the absence of splicing at the exon 4/intron 4 boundary . Cyclin D1 protein isoforms differ in the last 55 amino acids of the carboxy-terminus. Cyclin D1a and D1b proteins possess the cyclin box necessary for cyclin-dependent kinase (CDK) binding and enzymatic activity. But the PEST destruction box as well as Thr286, the phosphorylation site of glycogen synthase kinase-3β which promotes the nuclear export of cyclin D1 and its turn-over, are missing in form b [5, 6]. Recently, it has been shown that cyclin D1 isoforms could display functional differences and importantly that only cyclin D1b facilitates transformation in fibroblasts [7, 8, 9]. Considering this putative role in tumorigenesis, we have studied cyclin D1b expression in MCL and MM samples. A common A/G polymorphism described as a modulator of cancer risk is thought to regulate the production of cyclin D1b . We have also analyzed this relationship in MCL samples.
MCL and MM patients and cell lines
Patients with MCL or MM were diagnosed according to criteria from the World Health Organization classification in our institution (CHU Côte de Nacre, Caen, France); their clinical features are presented in the Additional file 1. Signed informed consent was obtained from all patients before all procedures as required by the institutional review board. Peripheral blood or bone marrow samples were obtained at diagnosis. Mononuclear cells were isolated by centrifugation over a Ficoll-Hypaque layer. CD19+ cells from 10 MCL patients were then purified (>90%) using a MACS microbeads system (Miltenyi Biotechnology). CD138+ cells from 6 MM patients were purified with the same system (purity >85%). JeKo-1, NCEB-1, HBL-2 and GRANTA-519 are MCL cell lines. Karpas 620, U266, NCI-H929, RPMI 8226, OPM-2 are MM cell lines. As previously reported by us, they all expressed cyclin D1 except OPM-2. Cell lines were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum and antibiotics.
Real-time quantitative RT-PCR
Sequences and positions of primers
QRT D1a S
5'-GGA AAG CTT CAT TCT CCT TGT TG-3'
QRT D1a AS
5'-TTC TTT TGC TTA AGT CAG AGA TGG AA-3'
QRT D1b S
5'-GCC AAT GGT CTG TGT GGT GAT-3'
QRT D1b AS
5'-ATT GGC CAC GCA CAT TGT G-3'
QRT 18S S
5'-CGG CTA CCA CAT CCA AGG AA-3'
QRT 18S AS
5'-GCT GGA ATT ACC GCG GCT-3'
5'-ATG TGA AGT TCA TTT CCA ATC C-3'
5'-CCA GTC AGT AAG TTC TAG GAG CAG-3'
Genomic DNA purification, cDNA synthesis and sequencing
Genomic DNA of CD19+ MCL cells was isolated with the Wizard® Genomic DNA (Promega). Total RNA was extracted from purified MCL cells and reverse-transcribed as before. Genomic DNA and cDNA were used as templates for PCR amplification using the Exon4 D1F and Intron4 D1R primers (see sequences in Table 1). PCR-amplified fragments encompassing the exon 4/intron 4 boundary were sequenced using the Big Dye Terminator Sequencing kit (Applied Biosystems). Sequences were analyzed with the ABI PRISM 310 Genetic Analyzer.
Nuclear, cytoplasmic or whole cell extracts, SDS-PAGE, electro-transfer and immunoblotting, all are procedures described previously . The DCS-6 monoclonal anti-cyclin D1 antibody (Ab, Pharmingen BD Biosciences n°556070) is directed against the entire cyclin D1 molecule and recognizes both isoforms; the sc-718 anti-cyclin D1 Ab (Santa Cruz Biotechnologies) is directed against the C-terminal part of the protein and is specific for cyclin D1a, the R3 Ab is directed against the C-terminal part of cyclin D1b and is specific for this isoform . The R3 Ab was a generous gift of Dr Alan J. Diehl. Reprobing blots with anti-β-tubulin Ab (sc-9104, Santa Cruz Biotech.) or anti-E2F1 (sc-251, Santa Cruz Biotech.) allowed us to check protein loading and transfer. Densitometric analyses were realized with a FluorSImager using the QuantityOne software (Bio-Rad).
Expression of cyclin D1 mRNA isoforms in MCL and MM patients and cell lines
Cyclin D1a and cyclin D1b mRNA expression in MCL and MM samples
CCND1 polymorphism, monoallelic transcription and cyclin D1 mRNA level
Expression, localization and stability of cyclin D1b protein in MCL samples
Analysis of CCND1polymorphism at position 9630 of genomic DNA and at position 870 of mRNA – Relation with the level of cyclin D1 forms a and b
CCND1 alternative splice could be modulated by a common G870A polymorphism within the splice donor site . Moreover, AA genotype could be associated with a preferential expression of isoform b and a higher risk of cancers . As presented in Table 3, MCL patient 5 was homozygous for the A allele, patient 6 homozygous for the G allele, the other 4 were heterozygous. Each tumor expressed either the A allele (3/6) or the G allele (3/6) but none expressed both alleles. Moreover, cyclin D1b was transcribed from the A or G allele and whatever the nucleotide at position 870, cyclin D1a was more expressed than cyclin D1b. The A/G genotype does not influence the relative level of the two transcripts.
Using standard RT-PCR, we found previously that cyclin D1a and b were coexpressed in MCL patients and in MM cell lines irrespectively of the presence of the t(11;14) translocation . We now extended this work and analyzed cyclin D1 isoform expression with a real-time quantitative RT-PCR in MM and MCL primary cells and cell lines.
Although cyclin D1 mRNA isoforms are always co-expressed in MCL and in cyclin D1-expressing MM cells, cyclin D1b protein was rarely and faintly expressed in MM samples. By contrast, in MCL cells, cyclin D1b was often present. Thus, post-transcriptional mechanisms regulate cyclin D1b status depending on the B-cell hemopathy.
Cyclin D1a and D1b proteins exhibit the same stability. In previous studies [7, 8], in transfected models, the calculated half-lives of cyclin D isoforms were also similar and similar to ours. Thus, endogenous and exogenous cyclin D1 isoforms are subjected to the same post-translational regulation. Either the PEST sequence is not strictly necessary for proteolysis through the proteasome machinery which is peculiarly active in MCL  or cyclin D1 can be degraded through another motif. In the case of cell response to stress, cyclin D1 can be degraded through its binding to the anaphase-promoting complex and a RXXL sequence located in the NH2-terminal part of the protein . This sequence is present in both cyclin D1 isoforms. Such a mechanism could be activated constitutively in MCL cells.
B-cell lymphomas can be induced in transgenic mice expressing a cyclin D1 mutant (T286A) under the control of Eμ enhancer . This mutant displays a 5-fold longer half-life than cyclin D1  and is exclusively nuclear. This sub-cellular localization seems to be a prerequisite to transformation. In MCL cells, cyclin D1b localizes both the nuclear and cytoplasmic compartments. In good agreement, endogenous cyclin D1b appears cytoplasmic and nuclear in oesophageal carcinomas  In contrast, in transfected fibroblastic cells, cyclin D1b remains mostly nuclear [7, 8, 9]. The simplest explanation is that the nuclear export is subverted by an overexpression of exogenous cyclin D1b.
Cyclin D1b is rarely and faintly expressed in MM and not always detected in MCL ( and our results). Thus, the expression of cyclin D1b is not necessary at least for the maintenance of a tumoral phenotype. Interestingly, the presence of a short cyclin D1 transcript (1.7 kb) lacking the 3'-UTR region responsible for mRNA stability, has been associated with MCL aggressiveness and a poor prognosis . The relationship between the alternative spliced forms and the short mRNA has to be studied.
In MCL, CCND1 alternative splicing does not modulate the level of transcripts b. This is consistent with results showing that CCND1 alternative splicing depends on sample origin . Moreover, cyclin D1b is transcribed independently of A/G genotype. This indicates that both alleles can splice to form both transcripts. Besides A/G polymorphism, trans-elements regulate alternative splicing. In good agreement with us, Howe and Lynas also showed that CCND1 polymorphism does not affect the prognosis of MCL patients .
In breast, sarcoma and colon cancers, with cyclin D1 overexpression and no chromosome 11 alterations, elevated mRNA levels result from a trans-acting influence of both alleles . A biallelic expression is seen in MM tumors without the t(11;14) . In agreement with that observed in MM tumors with t(11;14), only one allele is transcribed in MCL cells, the transcribed allele is likely the structurally altered one.
Although cyclin D1b mRNA is produced in the vast majority of MM and MCL cells, cyclin D1b protein is not always found and not found exclusively in the nucleus. These data allowed us to conclude that a transforming activity of cyclin D1b over cyclin D1a is unlikely. Moreover, our data establish no relationship between A/G polymorphism and cyclin D1b expression. The relevance of cyclin D1b in MCL and MM pathologies remains elusive.
The authors thank Anne Barbaras for expert technical assistance, Prof. Markus Müschen (Heinrich Heine Universität, Düsseldorf, Germany) for the gift of MCL cell lines, Dr Alan J. Diehl (University of Pennsylvania, Philadelphia, USA) for the gift of the R3 antibody. This work was supported by the Ligue Nationale contre le Cancer – Comité du Calvados, Cent pour sang la vie and the Association pour la Recherche contre le Cancer (grants n° 3426 and n° 7791 to BS). JG was the recipient of a scholarship from the Ministère de l'Enseignement Supérieur et de la Recherche.
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