Molecular Cloning And Ldentification Of The Retroviruses Associated With Human Leukemia

So far, the etiology and mechanisms of most human leukemias, which are common malignancies of hematopoietic tissues, remain unknown. Retroviruses have been suspected as causative agents of human leukemias for many years because of the definite etiological link between retroviruses and animal leukemias and human adult-T cell leukemia (ATL). However, numerous attempts to isolate and identify the putative retroviruses associated with these malignant diseases have not been successful to date. Our previous preliminary observations, together with other studies, suggest that, in addition to human adult-T cell leukemia, most other human leukemia may also be etiologically associated with unidentified human retroviral infection. The objective of this study is to identify the putative human retroviruses in primary leukemia cells from patients with various types of leukemia using a series of novel study strategies. Once the putative retroviruses are defined, innovative diagnostic, preventive and more efficient therapeutic strategies may be forthcoming.(I) Investigation of Retrovirai Particles and Viral Proteins in Human Leukemia CellsMaterials and MethodsIdentification of retroviral particlesThe morphology and ultrastructure of the viral particles were identified with electron microscopy.Identification of retroviral proteins in leukemic cells and normal blood cellsViral proteins in leukemia cells and normal blood cells were detected using immunocytochemical staining and identified with Western blot with retrovirus-specific monoclonal antibody p15E.Observation of cytopathologic effectCytopathologic effect of retroviruses on blood cells was assessed via observing syncytial cells under inverted microscope.Isolation and characterization of virus particles in primary leukemia cellsRetrovirus particles were generated in cultured leukemia cells in RPMI-1640 medium with fetal calf serum (PCS) plus hematopoietic growth factors and isolated from culture supernatants and purified in sucrose density gradient by centrifugation at 100,000g for 18hr at 4癈.Reverse transcriptase activity assayReverse transcriptase activity of retrovirus was assayed by radioisotope incorporation method.Analysis of viral protein profiles of retrovirus particlesProtein profiles of purified virus particles were analyzed by SDS-PAGE and identified with Western blot technique.ResultsIdentification of retrovirus particles in leukemic cells and normal blood cellsTo provide direct evidence of retroviral infection, the ultrathin sections of cultured cell samples from leukemia cases and healthy subjects were carefully examined for retrovirus particles by transmission electron microscopy. The results showed that typical retrovirus particles were observed in 20% (2/10) of fresh leukemia cell samples and all (20/20) of the cultured leukemic cell samples tested. In contrast, no retrovirus particles were found in 10 normal hematopoietic cell samples from healthy individuals. Based on the morphological properties and budding proceeding of the virus particles, these retrovirus particles belong to type C retroviruses and contain complete viral RNA genomes.Expression of retroviral proteins in leukemic cells and normal blood cellsTo search for the evidence of retroviral infection at protein level in leukemia patients, we evaluated the presence of retroviral proteins in leukemia cells from patients with various leukemias and normal blood cells from healthy donors using immunocytochemical staining with retrovirus-specific McAb p15E. Study results showed that retroviral proteins were detected in 80.65%(25/31) of fresh leukemic cell samples from 31 patients, but not in normal hematopoietic cell samples from 20 healthy donors. Western blot result revealed that there were two distinct protein bands (about 74KD and 24KD, respectively) in the protein extraction of leukemic cell specimens, which were strongly reacted with McAb p15E, but not in that of normal controls.Cytopathologic effects of retrovirus on b...