Our studies have shown that unconjugated antibody therapy targeted to
the B-cell marker CD20 by the anti-CD20 antibody, C2B8, inhibits the
growth of B-cell lymphomas in vitro. We have also demonstrated that
B-lymphoma tumor cells can be sensitized to subtoxic doses of
therapeutic drugs by the same antibody.
The objective of these studies is to delineate the mechanism by which
C2B8 alters B lymphoma cells, leading to inhibition of tumor cell
growth and sensitization to chemotherapeutic drugs. This study was
designed to examine: (1) whether C2B8 sensitizes the drug- and
toxin-resistant 2F7 cell line to tumor necrosis factor-alpha
(TNF-alpha), cisplatin (Platinol), fludarabine (Fludara), doxorubicin
(Adriamycin and others), and diphtheria toxin (DTX); (2) whether C2B8
regulates interleukin-10 (IL-10) secretion; (3) whether IL-10 is a
resistance factor in 2F7; and (4) whether C2B8 sensitization
regulates oncogenes and/or tumor-suppressor genes.
We demonstrate that C2B8 sensitizes 2F7 to doxorubicin, fludarabine,
TNF-alpha, and DTX-mediated cytotoxicity in a synergistic manner.
Furthermore, C2B8-targeted 2F7 downregulates IL-10 expression and
secretion after 24 and 72 hours of treatment.
Interleukin-10 has been shown to function as a resistance factor and
protects the tumor cells from the cytotoxic effects of therapeutic
drugs. We postulate that one method by which CD20 activation reverses
drug resistance involves IL-10 autocrine loops, which modulate
regulatory apoptotic proteins within the cell. This postulate is
verified by demonstrating that the addition of exogenous IL-10 to
C2B8-treated 2F7 caused a recovery of the cells, decreasing the
ability of C2B8 to sensitize.
We have also shown that C2B8 treatment downregulates the expression
of bcl-2 after only 4 hours. Involvement of bcl-2 could explain
resistance involved (and reversed) in an array of cytotoxic drugs.
Furthermore, C2B8 induces the upregulation of BTG-1 in 2F7 cells.
BTG-1 is a member of a family of proteins that induce cell cycle arrest.
CONCLUSION: The present findings demonstrate that the C2B8 antibody
exerts many activities on B-cell lymphoma, namely: (1) inhibition of
cell growth; (2) downregulation of the secretion of the protective
factor IL-10; (3) downregulation of the antiapoptotic Bcl-2 protein;
and (4) sensitization of lymphoma to several cytotoxic drugs through
IL-10 and Bcl-2 modulation.