Crosstalk Between Cyclins D1 and D3 in Mantle Cell Level At the Transcriptional and Postranscriptional Levels

Abstract 1373

Cyclin D1 (CCND1) expression is deregulated epigenetically in mantle cell lymphoma by the t(11;14) chromosome translocation. We have investigated the phenomenon of cyclin D1 mediated oncogene addiction in mantle cell lymphoma (MCL) and multiple myeloma (MM) cell lines. Gene targeting methods were utilized to generate Cyclin D1(-) MCL and MM cell lines. These cell lines did not make any detectable cyclin D1 mRNA and protein. These cells lines had shorter doubling times in vitro than their cyclin D1(+) counterparts and were also more chemoresistant in vitro and more tumorigenic in immunodeficient mice. Cyclin D3 mRNA and protein levels were increased dramatically. Q-RTPCR analysis showed a 17 fold increase in the mRNA of cyclin D3. The half-life of cyclin D3 protein was also significantly prolonged, from 20 minutes in wild type cells to 5 hours in mutant cyclin. Knockdown of cyclin D3 dramatically inhibited cell growth and caused apoptosis in the cyclin D1(-) but not in the t(11;14) cyclin D1 positive parental cells. Compensation and crosstalk between the cyclin D1 and D3 genes in MCL and MM cells were shown both in vitro and in vivo. Thus, the addiction to cyclin D1 in MCL and MM cells can be substituted by cyclin D3. Therapies that can target both cyclins D1 and D3 posttranscriptionally such as curcumin in combination with bortezomib were investigated in vitro in MCL cells. Combination treatment of MCL and MM cell lines with these agents produce significant downregulation of the protein levels of cyclins D1 and D3, even in cyclin D1(-) cell lines containing a extremely stable cyclin D3 protein.

In addition, we have shown that iron chelators such as desferoxamine can turn off cyclin D1 transcription and cause apoptosis of MCL cells in vitro.

Published work from several laboratories has described cyclin dependent kinase (Cdk) independent DNA binding activity for cyclin D1. Evidence that cyclin D1 and D3 might be involved in the pathogenesis of MCL in a cdk independent manner was observed.

We confirmed that cyclin D1 is a DNA binding protein and demonstrated by chromatin immuprecipitation assays that it binds to the endogenous cyclin D3 gene promoter both in a MCL cell line and in cells from leukemic MCL patients. The binding site corresponds to a published E2F binding site upstream of exon 2, which regulates two cyclin D3 transcript variants. We have also identified several potential targets of cyclin D1 regulation based on published cyclin D1 microarray experiments, several of which exhibit promoter- enrichment following cyclin D1 CHIP: GSK3b, ID3, and ANSN. Thus, cyclin D1 is a potential repressor of cyclin D3 expression by direct binding to the cyclin D3 promoter.

These data demonstrate crosstalk between cyclin D1 and D3 at both the transcriptional and post transcriptional levels. Furthermore, we demonstrate for the first time that cyclin D1 binds to the cyclin D3 promoter and potentially can mediate repression of cyclin D3 mRNA transcription. Deletion of cyclin D1 in an MCL cell line results in significant upregulation cyclin D3 mRNA and protein levels and a dramatic increase in the half life of cyclin D3 in cyclin D1 null cells. Previously reported data using knockdown and antisense technologies in MCL cell lines would be unable to appreciate these observations because of residual cyclin D1 mRNA and protein levels. Agents such as bortezomib and curcumin that downregulate cyclin D1 protein levels and iron chelating agents that downregulate cyclin D1 RNA levels may be useful agents in combination treatment of MCL and MM.

Figure 1:

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CHIP assay of cyclin D1 binding to the E2F binding site within the Cyclin D3 (CCND3) gene promoter in vitro and in vivo. Antibodies used include pan-H3 (Cell Signal #9715), Cyclin D1 (Abcam ab16663) and Normal Rabbit IgG (rb; Santa Cruz).

Figure 1:

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CHIP assay of cyclin D1 binding to the E2F binding site within the Cyclin D3 (CCND3) gene promoter in vitro and in vivo. Antibodies used include pan-H3 (Cell Signal #9715), Cyclin D1 (Abcam ab16663) and Normal Rabbit IgG (rb; Santa Cruz).

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