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Purpose: Arsenic, in the form of As(2)O(3), has gained therapeutic importance because it has been shown to be very effective clinically in the treatment of acute promyelocytic leukemia (APL). Via numerous pathways arsenic induces cellular alterations such as induction of apoptosis, inhibition of cellular proliferation, stimulation of differentiation, and inhibition of angiogenesis. Responses vary depending on cell type, dose and the form of arsenic. GSTO1, a member of the glutathione S-transferase superfamily omega, has recently been shown to be identical to the rate-limiting enzyme, monomethyl arsenous (MMA(V)) reductase which catalyzes methylarsonate (MMA(V)) to methylarsenous acid (MMA(III)) during arsenic biotransformation. In this study, we investigated whether arsenic trioxide (As(2)O(3)) induces apoptosis in both chemosensitive and chemoresistant cell lines that varied in their expression of p28 (gsto1), the mouse homolog of GSTO1., Methods: The cytotoxicity of arsenic in the gsto1- and bcl-2-expressing chemoresistant and radioresistant LY-ar mouse lymphoma cell line, was compared with that of the LY-ar's parental cell line, LY-as. LY-as cells are radiosensitive, apoptotically permissive, and do not express gsto1 or bcl-2. Cell survival, glutathione (GSH) levels, mitochondrial membrane potential, and stress-activated kinase status after arsenic treatment were examined in these cell lines., Results: As(2)O(3) induced an equivalent dose- and time-dependent increase in apoptosis in these cell lines. Cellular survival, as measured after a 24-h exposure, was also the same in each cell line. Reduced GSH was modulated in a similar time- and dose-dependent manner. Apoptosis was preceded by loss of mitochondrial membrane potential that triggered caspase-mediated pathways associated with apoptosis. With a prolonged exposure of As(2)O(3), both cell lines showed decreased activation of ERK family members, ERK1, ERK2 and ERK5. As(2)O(3) enhanced the death signals in LY-ar cells through a decrease in GSH, loss of mitochondrial membrane potential, and abatement of survival signals. This effect is similar to that seen when LY-ar cells are treated with thiol-depleting agents or by the removal of methionine and cysteine (GSH precursor) from the growth medium. This response is also completely contrary to that seen for radiation, actinomycin D, VP-16 and other agents, where LY-ar cells do not succumb to apoptosis., Conclusions: The overexpression of gsto1 in normally chemoresistant and radioresistant LY-ar cells renders them vulnerable to the cytotoxic effects of As(2)O(3), despite the 30-fold overexpression of the survival factor bcl-2. Gsto1 and its human homolog, GSTO1, may serve as a marker for arsenic sensitivity, particularly in cells that are resistant to other chemotherapeutic agents.

Purpose: To determine whether the difference in the apoptosis and clonogenic survival responses to radiation observed between the murine lymphoma cell lines LY-ar, which expresses bcl-2, and LY-as, which does not, was also evident after treatment with chemotherapy agents; and to determine whether clonogenic survival after chemotherapy agent exposure could be diminished by enhancing apoptosis through a decrease in cellular thiols., Methods: Cells were treated with cisplatin, VP-16, or Adriamycin, and apoptosis was determined using a DNA fragmentation assay. Cellular survival was quantified by limiting dilution assay. Intracellular thiols were decreased by maintaining LY-ar cells in cystine/methionine-free medium (CMF medium) for 7 h after drug treatment., Results: LY-as cells were approximately four times more likely to undergo apoptosis than LY-ar cells, having differences in apoptosis of 80% and 20%, respectively, for the agents used. LY-as cells were also more sensitive as measured by cellular survival, with a dose-modifying factor of about 1.8 measured at a 10% survival level. Incubation of LY-ar cells in CMF medium after drug treatment increased apoptosis and reduced clonogenic survival to the levels seen in LY-as cells, except after treatment with VP-16, where the reduction in cell survival was more modest., Conclusions: Decreasing intracellular thiols enhances apoptosis and cell killing in lymphoma cells after exposure to a variety of chemotherapy agents. This may be especially true for tumor cells that overexpress bcl-2, a gene that modifies cellular thiol status and conveys resistance to apoptosis. In this case, decreasing cellular thiols allows killing independent of the expression of bcl-2.

Purpose: Previously, we have reported that the bcl-2-expressing murine lymphoma cell line LY-ar is resistant to chemotherapy-induced apoptosis when compared to the non-bcl-2-expressing LY-as cell line. The intent of the present study was to determine whether this relationship extends to lymphomas produced from these cell lines in syngeneic mice, after treatment with the same chemotherapy agents., Methods: LY-ar and LY-as tumors were grown in the hind legs of syngeneic mice. They were subsequently exposed to graded doses of cisplatin (CP), etoposide (VP-16), Adriamycin (ADR), cytarabine (ara-C), cyclophosphamide (CY), or camptothecin (CAM). Apoptotic bodies were scored in histological sections of tumors that had been stained with hematoxylin and eosin. Tumor growth delay was determined on tumors that were treated when they were 8 mm in diameter. Thereafter, tumor diameter was measured daily with a vernier caliper until they had grown to a maximum of 16 mm in diameter., Results: When transplanted into host animals, tumors derived from these two cell lines and treated in vivo with CP, VP-16, ADR, ara-C, CY, and CAM displayed apoptotic propensities similar to those seen in the same cell lines when treated in vitro. Generally, for all the drugs tested, apoptotic indices in LY-as tumors were significantly higher than in LY-ar tumors. However, tumor growth delay measurements could not be predicted with any accuracy from the apoptotic indices. For some drugs LY-ar tumors were more sensitive than LY-as tumors (CP, Vp-16, ADR, ara-C), yet LY-ar tumors were more resistant to CY., Conclusions: Despite considerable interest in using apoptotic indices as predictors of treatment outcome, the data presented here suggest that these relationships are very complex. This may be especially true for chemotherapy agents for which effects in vivo are complicated by pharmacokinetics, host effects, and tumor cell heterogeneity.

Microscopic examination of histological sections of lymph nodes from a canine case of malignant lymphoma at 4 h after treatment with L-asparaginase revealed massive destruction of neoplastic cells by what was consistent with apoptosis morphologically. Apoptosis as the mode of cell death after asparaginase treatment was confirmed in a mouse lymphoma cell line (LY-TH) by the characteristic fragmentation of DNA into oligonucleosome-sized pieces and by the morphological changes consistent with apoptosis following treatment in vitro. Applied to these cells, asparaginase was found to be most cytotoxic over the range of 1-10 IU/ml. Even after 4 h of asparaginase treatment at 100 IU/ml, protein synthesis was reduced by only one-half, yet DNA fragmentation reached 40%. Other agents that affect protein synthesis (cycloheximide and actinomycin D) caused apoptosis as well; however, agents (radiation, prednisolone, and VP-16) whose mechanisms are different from inhibition of protein synthesis also caused apoptosis. As such, it seems unlikely that protein depletion per se and/or the elimination of specific short-lived proteins is the triggering event that leads to cell death. It is more likely that the suspension of cellular proliferation commits cells to apoptosis after asparaginase treatment.