Doctoral Dissertations

Date of Award

8-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Monica J. Justice

Abstract

Cancer is one of the leading causes of death in the human population. Effective treatment of cancer, as with any disease, depends upon the extent to which the etiology of the disease process is known. The main objective of this research is to further our understanding of the etiology of lymphoma by studying the molecular genetic basis of the development of lymphoma in mice. The identification of new loci that are implicated in causing murine lymphoma will facilitate our understanding of hematopoietic disease in humans. Retroviruses and other transposable elements are often found as causative agents in mouse cancers. One way these elements can cause cancer is to integrate near and alter the expression of normal somatic cellular proto-oncogenes. Retroviruses act as insertional mutagens and can serve as useful molecular tags to clone new genes causally associated with tumorigenesis. Nearly all strains that make up the AKXD recombinant inbred (RI) series of mice have a high incidence of lymphoma caused by the expression and subsequent somatic integration of murine leukemia retroviruses (MuLVs). The lymphomas of AKXD mice, therefore, can be used to identify and clone proto-oncogenes associated with lymphoma development using the MuLV sequence as a molecular tag. Conventional insertion site cloning requires the construction of genomic libraries which are screened by hybridization to isolate viral/genomic junction fragments. To eliminate the need for hybridization-based cloning, I adapted universal PGR to clone a viral insertion site in the mouse. Using a highly degenerate primer containing a short anchor sequence, coupled with a viral specific primer, we have amplified genomic sequences flanking a somatically acquired retrovirus in DNA isolated from a mouse B-cell lymphoma. This locus was designated lymphoid viral integration site 1, (Lvis1). Lvis1 was mapped to distal mouse chromosome 19 using interspecific backcross analysis. A detailed molecular genetic linkage analysis of this region, including a number of human gene markers known to map to human chromosome 10 revealed a significant region of conserved synteny between distal mouse chromosome 19 and human chromosome 10q23-q26. Analysis of Lvis1 in 270 AKXD tumors revealed that this locus is frequently altered by viral integration. Alterations at this locus have been observed in 9.6% of AKXD lymphomas, accounting for 22% of lymphomas classified as B-cell in origin. Notably, integrations at Lvis1 occur in a small number of T-cell tumors, but have not been observed in myeloid tumors. This locus is disrupted by retroviral integration in lymphomas from 14 AKXD strains. Random sequencing of BAC clones spanning >110 kb identified four genes near the site of viral insertion: Hex, mEg5, msec15, and L35a. Expression analysis of these genes in tumors with Lvis1 alterations revealed that viral insertions differentially effect neighboring gene regulation. Hex, an orphan homeobox gene involved in embryonic developmental patterning and hematopoiesis, is significantly upregulated in Lvis1 tumors, whereas no Hex expression is detected in tumors lacking Lvis1 alterations. Both mEg5 and msec15 show a slight upregulation in Lvis1 tumors, while L35a expression appears unaffected. These data provide the first genetic evidence for the possible role of these genes in lymphoma development, and highlight the use of genomic approaches for the study of position effect mutations.

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