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IMF 1996 Research Grant Award
By Melissa Alsina, University Of Texas
Abstract: Cloning of a novel osteoclast stimulatory factor (OSF) expressed by ARH-77 cells and bone marrow cells from myeloma patients in vivo. M. Alsina, R. Devlin, "S.V. Reddy," G.R. Mundy" and G.D. Roodman. VA Medical Center and University of Texas, San Antonio, Texas.

Osteolytic bone destruction is a very common manifestation of multiple myeloma (MM) and a major source of morbidity in this disease. However, in vivo mechanisms underlying the bone disease in MM are poorly understood. To study these mechanisms, we have developed a model of human myeloma bone disease in SCID mice. Mice injected with ARH-77 myeloma cells, (ARH-77 mice) developed diffuse osteopenia, lytic lesions, hypercalcemia, and osteoclastic bone resorption in areas of bone adjacent to myeloma cells. Although ARH-77 cells secrete IL-6 and lymphotoxin in vitro, elevated levels of these cytokines were not detected by a sensitive ELISA assay in marrow plasma from ARH-77 mice. Furthermore, conditioned media from ARH-77 and bone marrow plasma from ARH-77 mice induced bone resorption in bone organ cultures that was not blocked by anti-IL-6 or anti lymphotoxin antibodies. Therefore, we prepared an ARH-77 mammalian cDNA expression library to identify factors expressed by ARH-77 cells that stimulate osteoclast (OCL) activity. The library was screened by testing expression pools for the presence of factors that enhance OCL-like cell formation in mouse bone marrow cultures. Initial screening of the library identified an expression pool that stimulated formation of OCL-like cells in the absence of 1,25 (OH) 2D3. PCR screening of this expression pool for known bone-resorbing cytokines showed that it did not contain IL-6, IL-1a, IL-1B, TNF, annexin II or lymphotoxin cDNAs. We fractioned this pool to a single clone of 3.5kb and demonstrated that media from 293 cells transfected with this cDNA enhanced OCL-like cell formation. Sequence analysis of this clone showed it to be a novel gene with no homology of any known cytokine. RT-PCR analysis of marrow from 2 patients with MM as well as marrow ARH-77 mice demonstrated expression of this factor in vivo. Characterization of this novel OSF should provide important of the pathogenesis of bone destruction in patient with MM.

Six-Month Progress Report
I. Introduction Bone disease is a very common manifestation of multiple myeloma (MM) with 80% of the patients presenting with bone pain as one of its manifestations at diagnosis. Several known bone resorbing cytokines, such as IL-1, IL-6, and lymphotoxin have been implicated in the pathogenesis of myeloma bone disease, but their significance in vivo is not well defined. Although the factor(s) responsible for the bone destruction seen in MM in vivo have not yet been identified, it is known that there is an increase in osteoclast number and activity in areas of bone adjacent to myeloma cells along with decreased osteoblast number and new bone formation, resulting in uncoupling of the normal mechanisms of bone remodeling. This suggests that factor(s) secreted locally by the myeloma cells are responsible for the osteoclast activation and osteoblast inhibition. Since myeloma bone disease is a common problem for patients with MM, and a major source of morbidity, it is important to understand its pathogenesis. Such studies may lead to novel treatment alternatives for patients and improvement in quality of life and/or survival.

II. Preliminary Data

A. Myeloma bone disease in vivo model:

SCID immunodeficient mice injected with a human myeloma cell line (ARH-77 cells) developed lytic bone lesions by X-rays, hypercalcemia and osteoclastic bone resorption exclusively in areas of bone adjacent to myeloma cells. ARH-77 cell conditioned media and ARH-77 mice bone marrow plasma induced significant bone resorption in bone organ cultures when compared to control media and bone marrow plasma. This activity was not blocked by anti-IL-6 or anti-TNF§. Furthermore none of the known bone-resorbing cytokines was detected in the bone marrow plasma or bone marrow cells of the ARH-77 mice by ELISA or PCR, respectively.

B. Screening of an ARH-77 cDNA expression library for osteoclast activating factors:

We developed an ARH-77 cDNA expression library in order to identify the mediators of the bone resorption seen in our in vivo model. The library consists of 200 pools of 2000 clones each. Initial screening showed 6 pools that induced formation of osteoclast-like cells in human and mouse bone marrow cultures. Upon subcloning of pool #5 we identified a 3.5 kb clone (clone D24) with osteoclast formation activity in mouse bone marrow assays and expression in myeloma patients' bone marrow by PCR.

III. Ongoing Research

A. In vivo models: We have successfully transplanted SCID mice with cells from another human myeloma cell line IM-9. Mice developed hypercalcemia and lytic lesions by X-rays, after becoming paraplegic. The rate of engraftment is faster than ARH-77 cells.

B. Screening of the ARH-77 cell cDNA expression library:

We have completed the sequence of clone D24. It was determined to be a 2.8 kb clone with homology to the 3' region of TB1 gene, a gene that is overexpressed in colorectal carcinoma. Northern blot analysis suggests that the full length mRNA is 5 kb. It is expressed by both myeloma and normal bone marrow cells as well as several myeloma cell lines, including IM-9 cells, as assessed by PCR.

We have also continued screening the ARH-77 cDNA library for other novel osteoclast stimulatory factors. Another subpool from pool #5 (subpool 5.3) has marked activity in mouse bone marrow cultures, and we are in the process of subcloning this pool to a single clone.

IV. Future Experiments

A. Determine which known bone resorbing cytokines are secreted by IM-9 cells and if blocking the activity of the identified cytokines abolishes the bone-resorbing activity induced by IM-9 cells both in vitro and in vivo.

B. Determine if osteoclast stimulatory factors isolated from the screening of the ARH-77 cDNA expression library play a role in the myeloma bone disease in vivo models.

C. Clone the full-length DNA for D24 and confirm its activity as an osteoclast stimulatory factor, then determine its sequence and open reading frame. Once this is done, we will make an antibody against the clone to determine if it could block the bone-resorbing activity induced by ARH-77 cells and other myeloma cell lines in vitro and in vivo.

D. Determine if D24 is differentially expressed in myeloma versus normal patients by Northern Blot analysis.

E. Continue subcloning of other pools from the ARH-77 cDNA library that have osteoclast stimulatory activity in mouse and human bone marrow assays.

Editor's summary: The studies will attempt to identify new factors responsible for causing myeloma bone disease. With this knowledge, new treatment strategies can be developed.
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