Cyclophosphamide (CPA), a widely used oxazaphosphorine anti-
cancer prodrug, is inactive until it is metabolized by
cytochrome P450 to yield
phosphoramide mustard and
acrolein, which alkylate
DNA and
proteins, respectively.
Tumor cells transduced with the human
cytochrome P450 gene
CYP2B6 are greatly sensitized to CPA, however, the pathway of CPA-induced cell death is unknown. The present study investigates the cytotoxic events induced by CPA in 9L
gliosarcoma cells retrovirally transduced with
CYP2B6, or induced in wild-type 9L cells treated with
mafosfamide (MFA) or
4-hydroperoxyifosfamide (4OOH-IFA), chemically activated forms of CPA and its isomer
ifosfamide. CPA and MFA were both shown to effect
tumor cell death by stimulating apoptosis, as evidenced by the induction of plasma membrane blebbing, DNA fragmentation, and cleavage of the
caspase 3 and
caspase 7 substrate
poly(ADP-ribose) polymerase (PARP) in
drug-treated cells.
Caspase 9 was identified as the regulatory upstream
caspase activated in 9L cells treated with CPA, MFA, or 4OOH-IFA, implicating the mitochondrial apoptotic pathway in oxazaphosphorine-induced
tumor cell death. Correspondingly, expression of the mitochondrial proapoptotic factor Bax enhanced
caspase 9 activation, plasma membrane blebbing, and
drug-induced cytotoxicity. Conversely, overexpression of the mitochondrial antiapoptotic factor Bcl-2 blocked
caspase 9 activation, leading to an inhibition of
drug-induced plasma membrane permeability and blebbing,
terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity, PARP cleavage,
Annexin V positivity, and
drug-induced cell death. Although Bcl-2 thus blocked the cytotoxic effects of activated CPA, it did not inhibit the
drug's
cytostatic effects. CPA induced S-phase cell cycle arrest followed by conversion to an apoptotic pre-G1 state in wild-type 9L cells; by contrast, Bcl-2-expressing 9L cells accumulated in G2/M in response to CPA treatment. Intratumoral expression of Bcl-2 and related family members, including both apoptotic and antiapoptotic factors, is thus an important determinant of the responsiveness of
tumor cells to CPA and
ifosfamide, both in the context of conventional
chemotherapy and in patients sensitized to these oxazaphosphorine drugs by the use of
cytochrome P450-based gene therapy.