Molecular characterization of HTLV-I integration sites in patients with adult T-cell leukemia/lymphoma

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Mark B. Rabin - Committee Chair


Human T-cell lymphotropic virus type 1 (HTLV-I) is endemic for several areas of the world including central Africa, the southwestern islands of Japan, the Caribbean basin, and the southeastern United States. HTLV-I is etiologically associated with adult T-cell leukemia/lymphoma (ATL) based on several lines of evidence, including epidemiologic clustering of ATL in geographic regions endemic for HTLV-I infection, the high frequency of antiviral antibodies in the sera of ATL patients, detection of monoclonally-integrated provirus in the leukemic cells of ATL patients, and isolation of infectious virus from leukemic cell cultures. HTLV-I-induced leukemogenesis is postulated to result from a two-hit mechanism. Proviral DNA randomly integrates into the genome of a susceptible CD4$\sp+$ T-cell (first hit). Early infection is characterized by trans-activation of viral/host genes by the virally-encoded transcriptional control factor Tax. After a long latency period, a second step occurs to fix the leukemic phenotype. The precise nature of this second hit has not been defined, but may involve cis-activation of specific host genes by proviral insertion, host gene rearrangement, or host susceptibility factors. In an attempt to elucidate these secondary events, the work presented in this thesis focused on the cytogenetic and molecular characterization of proviral integration sites in primary tissue and cell lines derived from ATL patients. In situ hybridization was initially employed to map proviral integration sites in five ATL cell lines. Integration sites correlated with the map locations of oncogenic human genes and chromosomal loci associated with neoplastic disease (i.e., translocation breakpoints, chromosomal fragile sites) in all cases examined. Pulsed-field and standard Southern blot hybridization techniques were used to further define syntenic relationships between integrated provirus and candidate genes localized to viral integration sites. Northern analysis revealed no aberrant expression of selected candidate genes. To investigate integration sites in primary tumor tissue directly, a strategy called Alu-LTR PCR was developed which enabled cloning of human-specific viral flanking sequences. In each of three cases (two cell lines, one primary tumor), HTLV-I provirus integrated in close proximity to an Alu sequence, and the human-specific DNA between proviral and Alu sequences was A/T-enriched (69-75%). HTLV-I integrates at cytogenetically distinct loci in different tumors, but may preferentially target regions with an elevated A/T content.


Biology, Molecular; Biology, Genetics; Biology, Microbiology

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