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Fall 1998 Volume 3, Issue 2:
1998 Brian D. Novis Grant Progress Reports
Brian D. Novis Grant recipients are required to submit a six month progress report discussing their work. In the next issue of Myeloma Today we will include the report from our third 1998 recipient, Dr. Treon. In this issue, we present reports fro
Brian D. Novis Grant recipients are required to submit a six month progress report discussing their work. In the next issue of Myeloma Today we will include the report from our third 1998 recipient, Dr. Treon. In this issue, we present reports from our two other 1998 grant recipients.

Kaposi’s Sarcoma-Associated Herpesvirus Cytokines in the Oncogenesis of Multiple Myeloma

Principal Investigator: Matthew Rettig, MD
UCLA, Los Angeles, CA

The basic goal of this proposal is to characterize the biological effects of two KSHV derived cytokines (vIL-6 and vMIP-la) on human myeloma cells. In our proposal, we anticipated that the viral cytokines would retain the biological effects of the human homologues.

1. vIL-6 To date, we have focused on KSHV derived vIL-6. The vIL-6 protein was expressed in COS7 cells as a fusion protein with a hexahistidine residue. From the transiently transfected COS7 supernatants we purified vIL-6 on a nickel column. We titered the purified vIL-6 on B9 cells, and subsequently tested vIL-6 activity on proliferation (thymidine incorporation) and apoptosis (trypan blue dye exclusion and DNA fragmentation ladder) of human myeloma cell lines. The following are our results:

Although we demonstrated bioactivity of vIL-6 on B9 cells, our vIL-6 preparations had minimal proliferative affects (as measured by thymidine incorporation) on the AF-10 human myeloma cell line, yet AF-10 proliferated in response to human IL-6. Because the bioactivity of vIL-6 preps can vary, we studied three different preparations, and none demonstrated significant activity on AF-10.

We next evaluated the affects of vIL-6 on protecting human myeloma cells from apoptosis. We used the OCI-My5 cell line, because human IL-6 is known to inhibit apoptosis but not induce proliferation of this cell line. Quite interestingly, we showed that vIL-6 protects OCI-My5 cells from dexamethasone-induced apoptosis. In particular, there was a dose-response to the vIL-6 in terms of protection from apoptosis, with a 50% reduction in cell death at the highest concentrations of vIL-6 at 72 hours of dexamethasone exposure.

These initial data suggest that vIL-6 has a differential affect on induction of proliferation and inhibition of apoptosis of human myeloma cell lines. While KSHV derived vIL-6 did not induce proliferation of a human myeloma cell line, it did protect it from dexamethasone-induced apoptosis.

2. VMIP-1a : Using COS7 cells, we have expressed vMIP-la as a fusion protein with a hexahistidine residue at the amino terminus of the protein. We have purified our vMIP-la prep on a nickel column. Currently, we are optimizing our chemoattraction assay with recombinant human MIP-la before testing our viral preparation. For this purpose, we are using our bone marrow dendritic cell, RIT cell line, as the target for chemoattraction. We will also evaluate the ability of vMIP-la to upregulate human IL-6 in the RIT cell line.

The Role of Ras in Multiple Myeloma Plasma Cell Migration and Cell Surface Expression of RHAMM (Receptor for Hyaluronan Mediated Motility)

Principal Investigator: Anna Masellis, Ph.D.

Virginia Piper Cancer Institute,
Abbott North Western Hospital, Minneapolis, MN

We have previously identified the expression of RHAMM, the Receptor for Hyaluronan Mediated Motility, as a characteristic associated with MM cells grown in culture. The expression of RHAMM underlies the enhanced migratory behavior of myeloma plasma cells and circulating peripheral blood MM CD19+ B cells. (16). We have shown that RHAMM, expressed at the leading plasma membrane of migrating MM cells, co-localizes specifically with Ras, an oncogenic protein. We hypothesized that Ras activation and translocation to the plasma membrane was critical for RHAMM mediated MM cell migration.

To examine the effect of Ras on RHAMM-mediated migration of MM cells, we utilized the ANBL/6 cell line containing either wild-type N-Ras or N-Ras with an activating mutation at codon 12 (hereafter referred to as N-Ras12). The ANBL/6 cell line (provided by Dr. B. Van Ness, University of Minnesota) containing either wild-type N-Ras or N-Ras12, is non-motile and expresses low levels of cell surface RHAMM. An expression vector containing full length bone marrow (BM) RHAMM cDNA tagged with GFP (green fluorescent protein which allows detection of the RHAMM-GFP fusion protein by fluorescence microscopy) was introduced into the ANBL/6 cell lines. Using laser confocal microscopy, video time-lapse and FACS analysis, we determined whether mutations in N-Ras altered the distribution and function of RHAMM-GFP in the ANBL/6 cell line.

Our findings support our hypothesis of a role for Ras activation in RHAMM-mediated MM cell migration. We found that the transfection of full length MM BM RHAMM into ANBL/6 containing the wild-type N-Ras gene resulted in the cytoplasmic expression of RHAMM-GFP, with no increase in ligand binding or cell migration compared to mock transfected ANBL/6 controls cells. In contrast, transfection of full length MM BM RHAMM cDNA into ANBL/6 containing N-Ras 12 resulted in the increased plasma membrane expression of RHAMM-GFP, increased ligand binding and increased cell migration. Thus, the presence of an activated N-Ras converted the non-motile ANBL/6 cells to a phenotype corresponding to that seen with circulating MM plasma cells studied in vitro.

Masellis, Anna M., Belch, Andrew R., Van Ness, B., Mant, Michael M. and Pilarski, Linda M. A central role for the Ras oncogene in RHAMM-mediated spread of myeloma. 1998

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