Effects of Newcastle Disease Virus Strains Af 2240 and V4-Upm on Cytolysis and Apoptosis of Leukemia Cell Lines
Newcastle disease virus (NDV) is a member of the Paramyxoviridae that has caused severe economic losses in the poultry industry worldwide. Several strains of NDV were reported to induce cytolysis to cancerous cell lines. In this study, two NDV isolates namely, AF 2240 and V4-UPM were evaluated fo...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2008
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/7569/1/ABS_-----__FBSB_2008_20.pdf |
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| Summary: | Newcastle disease virus (NDV) is a member of the Paramyxoviridae that has caused
severe economic losses in the poultry industry worldwide. Several strains of NDV
were reported to induce cytolysis to cancerous cell lines. In this study, two NDV
isolates namely, AF 2240 and V4-UPM were evaluated for their anti-leukemic
properties against four leukemic cell lines - HL60 (Promyelocytic leukemia), WEHI
3B (Mouse myelomoncytic leukemia), CEMSS (Human T-lymphobalstic leukemia)
and K562 (Erythromyelobalstic leukemia). The cytolytic effects of NDV strains AF
2240 and V4-UPM towards WEHI 3B, HL60, CEMSS and K562 cell lines were
determined using microtetrazolium (MTT) assay. The cytolytic dose - fifty percent
(CD50) for WEHI 3B, HL60 and CEMSS treated with AF 2240 strain were 2, 25, 16
HAU, respectively, while the CD50 for WEHI 3B, HL60 and CEMSS treated with
V4-UPM were 8, 110 and 64 HAU, respectively.
Comparatively, both NDV strains showed very low cytolytic activity against K562
and non-leukemic cell lines namely, 3T3 (mouse fibroblasts), mouse lymphocytes and human peripheral lymphocytes. Further studies were done to observe the
morphological changes in the WEHI 3B treated cells using light, transmission and
scanning electron microscopes. The apoptosis and necrosis were examined under
fluorescence microscope, where the cells were stained with acridine orange (AO) and
propidiun iodide (PI). The treated cells with NDV strains AF 2240 and V4-UPM
showed apoptotic features such as cell shrinkage, cell blebbing, and formation of
apoptotic bodies compared to the control cells that did not express any features for
apoptosis and necrosis. The early apoptosis was also observed under fluorescence
microscope, where the cells were stained with Annexin V and PI.
The virus effect on cell proliferation was determined by MTT assay and BrdU
techniques. Furthermore, at molecular level, both NDV strains caused
internucleosomal DNA cleavage producing a multiple of 180-200 bp fragments, that
were visible as a ladder on the agarose gel. Early apoptosis was also observed using
Annexin V flow cytometry method. The percentage of apoptosis for WEHI 3B cells
treated with NDV strains AF 2240 and V4-UPM had increased with time. Cell cycle
and apoptosis were also determined using flow cytometry PI method. Both NDV
strains were not able to arrest WEHI 3B at specific cell cycle phases using flow
cytometry PI method.
In this study anti-leukemic activity of both NDV strains was evaluated on BALB/c
mice induced leukemia with WEHI 3B cells. A day later they were treated with NDV
strains AF 2240 and V4-UPM and arabinocytocine, a commercial drug, as positive
control. The mice groups treated with arabinocytocine, NDV strains AF 2240 and
V4-UPM showed significant killings (p<0.05) of leukemic cells compared to the mice group without any treatment. The total white blood cell and percentage of
blasts cell in the blood, bone marrow and spleen smears were significantly low
(p<0.05) in mice treated with the arabinocytocine, NDV strains AF 2240 and V4-
UPM compared to the mice group without treatment, that showed high number of
leukemia. Spleen and liver weights were significantly low (p<0.05) in the mice
groups treated with arabinocytocine, NDV strains AF 2240 and V4-UPM compared
to the mice group without treatment, that showed significant (p<0.05) splenomegaly
and hepatomegaly.
Histopathological studies carried out had confirmed haematological results. From the
results obtained, the mice groups treated with both NDV stains AF 2240 and V4-
UPM showed similar results as arabinocytocine, which is a commercial drug for
leukemia. Immunoperoxidase staining, haemaggulatintion test and real time PCR
were carried out to detect NDV in mice organs after treatment with NDV. The results
showed no NDV particles were detected in the organs. This study showed that NDV
strains AF 2240 and V4-UPM had caused cytolytic effects against WEHI 3B
leukemia cell line in vitro and in vivo. |
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