Induction of Cyclin D1 by HGF, IGF-1 and IL-6 in a Human Myeloma Cell Line with a t(11:14) Translocation.

Almost all patients with multiple myeloma have dysregulation of cyclin D1 -2 or -3. The t(11:14) translocation juxtapose the CCND1 locus coding for the cyclin D1 protein to the IgH locus. This leads to upregulation of the cyclin D1 gene, and thus to increased proliferative capacity. Despite the presence of IgH translocations, the myeloma cells are highly dependent of the bone marrow microenvironment for malignant growth. Whether the expression of cyclin Ds is additionally regulated by the microenvironment needs to be further examined. Here we report a myeloma cell line, INA-6, with a t(11;14) translocation in which expression of cyclin D1 is regulated by several cytokines. INA-6 cells were analysed by interphase FISH with probes matching the CCND1 gene (coding for the cyclin D1 protein) and the Ig heavy chain locus. We found co-localisation of the two probes indicating a translocation between chromosome 14 and chromosome 11. We detected a total of five signals for CCND1, indicating the occurrence of three extra copies of chromosome 11. This was confirmed by an analysis with a single probe detecting the CCND1 locus. We further analysed the content of cyclin D1 protein by western blotting after the cells had been washed and grown in serum free media with or without cytokines for 24 hours. INA-6 constitutively expressed cyclin D1. HGF, IL-6 and IGF-1 further enhanced the expression of cyclin D1. In combination with IL-6, HGF had a synergistic effect on cyclin D1 expression. The constitutive cyclin D1 expression is probably caused by the translocation between chromosome 11 and 14. However the increase in number of cyclin D1 genes not co-localized to the IgH chain can also contribute to the basal level of cyclin D1 expression in this cell line as well as post-transcriptional regulation. The increase in cyclin D1 expression after cytokine stimulation can be explained by an increase in transcription from the normal CCND1 copy or the other extra copies. One intriguing possibility is that the translocation itself makes the cell susceptible to cytokine regulation of cyclin D1. At least IGF-1 and IL-6 are known for their ability to enhance immunoglobulin production and may therefore regulate cyclin D1 through the IgH promotor in the t(11:14) translocation.

The different ways to induce cyclin D1 protein in this cell line is a functional example of the collaboration between the dysregulated myeloma cell and the components of its microenvironment in the bone marrow in regulating a key component of cell cycling and a possible oncogene in multiple myeloma.