Epstein-Barr virus (EBV)-infected
B cell lymphomas are resistant to apoptosis during
cancer development and treatment with
therapies. The molecular controls that determine why
EBV infection causes apoptosis resistance need further definition. EBV-positive and EBV-negative BJA-B
B cell lymphoma cell lines were used to compare the expression of selected apoptosis-regulating Bcl-2 and
caspase proteins in EBV-related apoptosis resistance, after 8 hr or 18-24 hr
etoposide treatment (80 microM). Apoptosis was quantified using morphology and verified with
Hoechst 33258 nuclear
stain and electron microscopy. Fluorescence activated cell sorting (FACS) was used to analyse effects on cell cycle of the
EBV infection as well as
etoposide treatment. Anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bax,
caspase-3 and
caspase-9 expression and activation were analysed using Western immunoblots and densitometry. EBV-positive cultures had significantly lower levels of apoptosis in untreated and
etoposide-treated cultures in comparison with EBV-negative cultures (p < 0.05). FACS analysis indicated a strong G2/M block in both cell sublines after
etoposide treatment. Endogenous Bcl-2 was minimal in the EBV-negative cells in comparison with strong expression in EBV-positive cells. These levels did not alter with
etoposide treatment. Bcl-XL was expressed endogenously in both cell lines and had reduced expression in EBV-negative cells after
etoposide treatment. Bax showed no
etoposide-induced alterations in expression.
Pro-caspase-9 and -3 were seen in both EBV-positive and -negative cells.
Etoposide induced cleavage of
caspase-9 in both cell lines, with the EBV-positive cells having proportionally less cleavage product, in agreement with their lower levels of apoptosis.
Caspase-3 cleavage occurred in the EBV-negative
etoposide-treated cells but not in the EBV-positive cells. The results indicate that apoptosis resistance in EBV-infected
B cell lymphomas is promoted by an inactive
caspase-3 pathway and elevated expression of Bcl-2 that is not altered by
etoposide drug treatment.