Relevance of cyclin D1b expression and CCND1polymorphism in the pathogenesis of multiple myeloma and mantle cell lymphoma

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.

Total RNA was isolated from purified MCL and MM cells and cell lines with the RNeasy Micro Kit (QIAGEN S.A.) and reverse-transcribed with the 1^st Strand cDNA Synthesis kit (Roche). Real-time quantitative RT-PCR was performed on an ABI PRISM 7700 Sequence Detector with the SYBR^® Green PCR Master mix (Applied Biosystems). Primers were designed with the ABI PRISM Primer Express^® software; their sequences are presented in Table 1. For MCL and MM samples, RT-PCR amplifications were performed in a final volume of 15 μl containing PCR master mix, 5 ng of cDNA equivalents and primers (300 nM each for 18S rRNA, 400 nM for cyclin D1a and 800 nM for cyclin D1b). As negative control, real-time PCR was performed with purified non reverse-transcribed RNA of each sample. Expression of cyclin D1 isoforms compared to 18S rRNA was evaluated with the comparative threshold method (Ct). Each experiment was done in triplicate, triplicate values were averaged and averaged Ct values for 18S rRNA were subtracted from each value to give the ΔCt. U266 and JeKo-1 cells expressing high levels of both cyclin D1a and D1b mRNA were chosen as calibrators for MM and MCL cells, respectively. ΔCt obtained from U266 or JeKo-1 was subtracted from the ΔCt of each cell line to give ΔΔCt. The relative amount of each cyclin D1 isoform compared to the calibrator was calculated by the formula N = 2^-ΔΔCt.

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 [10]. 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 [7]. 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).

Both spliced mRNAs have been detected by standard RT-PCR in MCL primary cells and MM cell lines [11]. To quantify the relative amounts of mRNA isoforms, primers specific for each isoform were designed and real-time quantitative RT-PCR was performed. The reliability of the method is illustrated in Figure 1. Total RNA purified from U266 cells was reverse-transcribed and serially diluted and samples were then amplified using the specific primers. A linear correlation was confirmed by the plot of input amount of RNA (X-axis) vs. Ct values (Y-axis) with minimal deviation among duplicates or triplicates. The efficacy of amplification was 100% for cyclin D1a and cyclin D1b allowing the comparison of their relative levels. The expression of cyclin D1 transcripts was normalized to 18S rRNA as internal standard. As expected, cyclin D1a mRNA was detected in all MCL patients with ΔCta varying from 8.8 to 12.9 (Table 2). In 9 out of 10 MCL patients, we found the co-expression of isoform b with ΔCtb varying from 14.3 to 19.3. MCL patient 3 expressed only the isoform a. Cyclin D1a was predominant over cyclin D1b with a ratio R varying from 33 to 80 (Table 3). In MCL cell lines, isoform levels were almost in the same range than in primary cells (ΔCta 6.5–12.5, ΔCtb 12.7–19.7). In only 3 out of 6 MM patients, total cyclin D1 was detected in CD138+ purified cells (data not shown); these three patients were studied further for cyclin D1 isoform expression. As expected, in MM primary cells and cell lines, cyclin D1 a and b expression was highly heterogeneous (ΔCta 10.0–18.2, ΔCtb 18.5–27.7) but perfectly correlated. They globally expressed less cyclin D1b mRNA than MCL (Table 2). The calculation of N illustrates the heterogeneity of cyclin D1a and b expression inside each series of samples (MM vs. MCL) and the heterogeneity of cyclin D1a vs. cyclin D1b expression for each case (see Additional file 2).

We detected the presence of both protein isoforms in MCL samples except for patient 3 (Figure 2A) and in cell lines (Figure 2B). The protein level of cyclin D1a (estimated by densitometric analysis) was higher than of form b (from 2 to 15-fold). In whole cell extracts, cyclin D1b was not detected in MM cell lines (data not shown), in good agreement with previous report [11]. Since cyclin D1 isoforms could display different sub-cellular localizations, nuclear and cytoplasmic protein extracts were prepared from MCL cell lines and primary cells and from MM cell lines, then immunoblotted. In MCL cell lines and primary cells, cyclin D1a (data not shown) and cyclin D1b localized both the nuclear and the cytoplasmic compartments with the exception of Pt 2 for which cyclin D1b was only nuclear (Figure 3). A very faint band was also visible in both extracts in NCI-H929 MM cells. Although not having the nuclear export signal, cyclin D1b displays the same sub-cellular distribution than cyclin D1a. GRANTA-519 and JeKo-1 cell lines were treated with cycloheximide to inhibit protein synthesis. We then monitored protein stability at the times indicated in the Figure 4. Cyclin D1b exhibited a half-life similar to cyclin D1a in both cell lines (indicated under each immunoblot). Both are short-lived proteins despite the absence of PEST sequences in isoform b.

CCND1 alternative splice could be modulated by a common G870A polymorphism within the splice donor site [4]. Moreover, AA genotype could be associated with a preferential expression of isoform b and a higher risk of cancers [12]. 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.