Detection of drug-induced apoptosis and necrosis in human cervical carcinoma cells using 1H NMR spectroscopy.

Apoptosis and necrosis need to be differentiated in order to distinguish drug-induced cell death from spontaneous cell death due to hypoxia. The ability to differentiate between these two modes of cell death, especially at an early stage in the process, could have a significant impact on accessing the outcome of anticancer drug therapy in the clinic. Nuclear magnetic resonance spectroscopy was used to distinguish apoptosis from necrosis in human cervical carcinoma (HeLa) cells. Apoptosis was induced by treatment with the topoisomerase II inhibitor etoposide, whereas necrosis was induced by the use of ethacrynic acid or cytochalasin B. We found that the intensity of the methylene resonance increases significantly as early as 6 h after the onset of apoptosis, but that no such changes occur during necrosis. The spectral intensity ratio of the methylene to methyl resonances also shows a high correlation with the percentage of apoptotic cells in the sample (r2=0.965, P<0.003).

Expression of p53 predicts treatment failure in aggressive non-Hodgkin's lymphomas.

Approximately 50% of patients with aggressive non-Hodgkin's lymphomas (NHL) achieve a complete remission (CR) and cure with combination chemotherapy. The International Index is a useful clinical measure that predicts the patients' tolerance of therapy and likelihood of achieving CR, but it is not a direct measure of chemosensitivity. In this study we have investigated the predictive value of the tumor suppressor gene, p53, as a biological marker for response to treatment in the aggressive NHL. A retrospective study was carried out on 50 patients with aggressive NHL who were treated with CHOP chemotherapy. Treatment outcome was correlated with the expression of p53 in the lymphoma, as measured by routine immunohistochemistry using the monoclonal antibody Do-7. Forty percent of the lymphomas had >5% of the cells staining positively for p53 and this finding correlated significantly with response to treatment. Fifty percent of patients with p53 positive tumors achieved a CR versus 77% of patients with p53 negative tumors. In addition, the relapse rate and time to relapse were significantly different in the two groups. In the p53 positive group, 60% of patients relapsed in a median time of 6 months, whereas 26% of the p53 negative group relapsed with the time to relapse being >22 months. The overall survival of the p53 positive group (17 months) was significantly shorter than that of p53 negative group (>24 months). These results suggest that the immunohistochemical assessment of p53 is a simple and important prognostic measure for patients with aggressive NHL who are treated with CHOP chemotherapy.

P53, MDM-2, BAX and BCL-2 and drug resistance in chronic lymphocytic leukemia.

Most antitumor agents exert their cytotoxic effect through the induction of apoptosis, and this process may be mediated through an elevation in p53 protein, with a subsequent increase in bax and decrease in bcl-2. p53 also increases mdm-2 expression and mdm-2 may then bind and inactivate p53. Cells from 31 patients with chronic lymphocytic leukemia (CLL) were treated in vitro with 2-chlorodeoxyadenosine (CdA), arabinosyl-2-fluoroadenine (F-ara-A), or chlorambucil (CLB) and drug sensitivity measured using the MTT assay. The protein levels of bax and bcl-2 were measured in CLL cells from 25 patients, and were found to be higher in leukemic cells than in normal B cells. The bcl-2 levels varied three-fold, the bax levels fifteen-fold, and the bax:bcl-2 ratios ranged from 0.44 to 2.91. The expression of mdm-2 mRNA was measured in CLL cells from 28 patients and was found to vary twenty-fold. However, no correlation was observed between drug sensitivity to CdA, F-ara-A, or CLB and the cellular levels of mdm-2 mRNA, or the protein levels of bax or bcl-2, or the bax:bcl-2 ratio. Treatment of CLL cells having wild type p53 with CdA, F-ara-A or CLB produced an increase in p53 protein and mdm-2 mRNA. This was not observed in cells having a p53 mutation, and these cells were highly resistant to both CLB and the nucleoside analogs. In contrast to the nucleoside analogs and CLB, dexamethasone and vincristine had no effect on mdm-2 mRNA levels. Treatment of CLL cells containing a wild type p53 gene with CdA, F-ara-A, or CLB, did not produce any consistent changes in bax or bcl-2. Thus, CdA, F-ara-A and CLB appear to act in CLL cells through a p53-dependent pathway, whereas this does not occur with dexamethasone or vincristine. The cellular levels of mdm-2, bcl-2, bax or the bax:bcl-2 ratios are not predictive indicators of clinical sensitivity in CLL, but an increase in mdm-2 levels after drug treatment is indicative of p53 function in these cells.

In vitro cytotoxicity of 2-chlorodeoxyadenosine and chlorambucil in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is most commonly treated with the alkylating agent chlorambucil (CLB), although the nucleoside analogs, fludarabine (Flu) and 2-chlorodeoxyadenosine (CdA), are also effective in this disease. In this study, we investigated the in vitro cytotoxicity of CdA and CLB in CLL cells from 12 patients in vitro. Treatment with CLB for 6 h, followed by CdA for 18 h, resulted in 2.3- to 7.5-fold synergistic cytotoxicity in leukemic cells from 10 patients and an additive effect in cells from two patients. In general, synergy was greatest in patients who were sensitive to CLB or CdA, and could be enhanced by increasing the concentrations of CLB or CdA. Synergy was only observed if the cells were treated with CLB prior to CdA. Synergy could not be explained by an increase in the incorporation of CdA into DNA, or by the inhibition of repair of CLB-induced DNA crosslinks by CdA. In contrast to CLL cells, treatment of human marrow in vitro with CLB and CdA resulted in a low level of synergy for CFU-GM cells, and additive cell kill in erythroid progenitors. Thus, treatment with CdA and CLB can produce selective synergistic cell kill in CLL cells, and combination therapy may improve the therapeutic index of these agents in chemosensitive patients.

Comparison of antitumor activities of 2-chlorodeoxyadenosine and 9-beta-arabinosyl-2-fluoroadenine in chronic lymphocytic leukemia and marrow cells in vitro.

The in vitro antitumor activities of the nucleoside analogs, 2-chlorodeoxyadenosine (CdA) and 9-beta-arabinosyl-2-fluoroadenine monophosphate (Flu), and the alkylating agent, chlorambucil (CLB), were compared in leukemic cells from 28 patients with chronic lymphocytic leukemia (CLL). On a molar basis, the median sensitivities of the cells to these agents were CLB > CdA > Flu. CLL cells from 90% of the patients had similar relative orders of sensitivities to CdA and Flu, while cells from 10% of the patients showed differential sensitivities to these agents. There was no relationship between the sensitivities of the cells to the nucleoside analogs and sensitivity to CLB. CdA and CLB produced similar toxicities to human marrow progenitor cells in vitro, while Flu was less toxic to these cells. An 18 h exposure to CdA produced significantly greater cell kill of both CLL and marrow progenitor cells than an equivalent 2 h treatment; however, the difference in cytotoxicity was greater for the tumor cells resulting in a higher therapeutic index with the 18 h treatment. The intracellular accumulation of drug varied 5-fold for CdA, with the major metabolite being CdAMP, and 15-fold for Flu, with the major metabolite being F-ara-ATP. However, the accumulation of CdA, Flu or their metabolites did not predict for drug sensitivity. These studies suggest that CdA and Flu cross-resistance cannot be assumed in all CLL patients. The therapeutic effectiveness of CdA may be enhanced by use of a prolonged, low-dose drug regimen.

Chlorambucil induced apoptosis in chronic lymphocytic leukemia (CLL) and its relationship to clinical efficacy.

Chlorambucil induced apoptosis was measured in CLL cells treated with clinically achievable drug doses in vitro. While spontaneous apoptosis occurred in CLL cells incubated in vitro in the absence of drug, the level of apoptosis, as measured by the extent of DNA fragmentation, was greater in cells treated with chlorambucil. In addition, macrophages were shown to engulf drug treated CLL cells in vitro. To determine if chlorambucil can also induce apoptosis in vivo, CLL cells were isolated from patients before treatment and at intervals after clinical therapy with chlorambucil (0.9 mg/kg given over 3 days). Apoptosis was measured in these cells immediately after isolation and following incubation in vitro for 72 hr. No apoptotic changes were detected in cells immediately after isolation either before or after clinical treatment. In contrast, apoptosis was observed in cells that were incubated ex vivo for 72 hr, and the level of apoptosis was greater in cells that were isolated after chlorambucil treatment compared with cells obtained prior to therapy. The increased apoptosis observed in CLL cells ex vivo after therapy was related to the fall in the patient's lymphocyte count. In general, a large increase in apoptosis ex vivo after treatment was followed by a significant decrease in the patient's lymphocyte count. Thus, chlorambucil may produce its antitumor effect in CLL by inducing apoptosis-associated membrane changes that result in rapid clearance of the apoptotic cells by the immune system.

Role of transforming growth factor-beta in chronic lymphocytic leukemia.

TGF-beta is an important immunoregulator as it suppresses proliferation and function of B- and T-lymphocytes. In the present study we have examined the cellular localization and secretion of TGF-beta in B-cells from normal donors and patients with CLL and have assessed the influence of TGF-beta 1 on DNA synthesis in these cells. Using anti-LC(1-30)--a polyclonal anti-TGF-beta 1 antibody--TGF-beta was localized to discrete sites within the cytoplasm of both normal and malignant lymphocytes. These areas co-localized with areas detected by an antigranule antibody (D545), suggesting that TGF-beta may be stored within cytoplasmic secretory vesicles. Both normal B- and CLL cells contained low or undetectable levels of TGF-beta mRNA and secreted low and equivalent amounts of TGF-beta. Compared to untreated cells, DNA synthesis was reduced by TGF-beta 1 to a mean +/- S. E. of 0.84 +/- 0.07 in CLL cells and this was significantly less (p < 0.001) than that observed in normal B-cells (mean +/- S. E. of control, 0.12 +/- 0.02). In 3 of the 18 patients, TGF-beta 1 stimulated DNA synthesis. The reduced inhibition of leukemic cell DNA synthesis by TGF-beta 1 in CLL may provide these cells with a growth or survival advantage over normal lymphocytes and contribute to their selective accumulation.

Induction of apoptosis in CD4+ prolymphocytic leukemia by deoxyadenosine and 2'-deoxycoformycin.

The leukemic cells of a patient with CD4+ prolymphocytic leukemia were treated in vitro with 5 microM deoxyadenosine and 60 microM 2'-deoxycoformycin (dCF), an inhibitor of adenosine deaminase (ADA). Following treatment, the leukemic cell dATP level increased to 378 pmol/10(6) cells on day 3, after which the level plateaued. Apoptosis was apparent following 4 h of incubation, and by day 8 34% of the chromatin was fragmented. Apoptosis also occurred in control cells, but to a lesser extent than in drug-treated cells. When the patient was treated with dCF, 4 mg/M2 i.v. the leukemic cell ADA activity was inhibited 24 h following treatment, and the lymphocyte dATP content increased to 303 pmol/10(6) cells by day 6. The lymphocyte count fell 60% in 1 week, but during this time there was no evidence of apoptosis in these cells. Thus, if dCF induces apoptosis in vivo, the effete cells may be rapidly cleared from the circulation and thus elude detection.

Role of glutathione and glutathione S-transferase in chlorambucil resistance.

A chlorambucil (CLB)-resistant cell line, N50-4, was developed from the established mouse fibroblast cell line NIH 3T3, by multistep drug selection. The mutant cells exhibited greater than 10-fold resistance to CLB. Alterations in GSH and glutathione S-transferase (GST) were found in CLB-resistant variants. A 7-10-fold increase in cellular GSH content and a 3-fold increase in GST activity were detected in N50-4 cells, compared with parental cells, as determined by enzymatic assays. An increase in steady state levels of the GST-alpha isozyme mRNA was found in the CLB-resistant cells, as analyzed by Northern blotting. No GST gene amplification or rearrangement was shown by Southern blot analysis. To test the relative roles of GSH and GST in CLB resistance, a number of GSH- and GST-blocking agents were used. The CLB toxicity was significantly enhanced in N50-4 cells by administration of either the GSH-depleting agent buthionine sulfoximine or the GST inhibitors ethacrynic acid or indomethacin. The resistance to CLB cytotoxicity in N50-4 cells, however, was still significantly higher than that of parental cells. The resistance of N50-4 cells to CLB was almost completely abolished by combination pretreatment yielding both GSH depletion and GST inhibition. The results indicate that both increased cellular GSH content and increased GST activity play major roles in CLB resistance in N50-4 mutant cells.

Mechanisms of resistance to chlorambucil in chronic lymphocytic leukemia.

The postulated biochemical mechanisms responsible for clinical resistance to chlorambucil (CLB) in chronic lymphocytic leukemia (CLL) have been examined. The total sulfhydryl, non-protein-bound sulfhydryl, protein-bound sulfhydryl (PSH) and glutathione (GSH) levels, in addition to glutathione S-transferase (GST) activities, were measured in the leukemic cells of 18 CLL patients. In addition, the formation and repair of DNA cross-links were measured following incubation of the cells with 100 microM chlorambucil in vitro. These parameters were then correlated with the subsequent clinical responses of the patients, as measured by the percent fall in lymphocyte count 3 weeks following 0.9 mg/kg chlorambucil. No correlations were observed between any of the individual parameters and clinical response, although a slight positive correlation was observed between the PSH:GSH ratio and clinical response. These findings suggest that multiple mechanisms may contribute to CLB-resistance in CLL.

Glutathione S-transferase activity, sulfhydryl group and glutathione levels, and DNA cross-linking activity with chlorambucil in chronic lymphocytic leukemia.

Glutathione (GSH) levels and glutathione S-transferase (GST) activities were measured in the leukemia cells of 12 patients with chronic lymphocytic leukemia. Both were correlated with prior clinical exposure to alkylating agents and with DNA cross-link formation by chlorambucil in these cells in vitro. No correlation was observed between prior exposure to alkylating agents and GSH level or GST activity. An inverse correlation was observed between GST activity and cross-linking by chlorambucil, which was enhanced if both GST activity and GSH level were related to cross-linking. These findings suggest that the combination of GST and GSH protects the DNA of leukemia cells from chlorambucil, but the role of this combination in clinical resistance remains to be determined.

Repair of gamma-ray induced DNA strand breaks in the radiation-sensitive mutant rad18-2 of Saccharomyces cerevisiae.

The repair of gamma-ray induced DNA single and double-strand breaks was looked at in wild type and rad18-2 strains of the yeast Saccharomyces cerevisiae using sucrose gradient centrifugation. It was found that rad18-2 diploid cells could repair single and double-strand breaks induced by gamma-rays. It was also found that rad18-2 cells experienced a breakup of their DNA during post-irradiation incubation to a size smaller than seen in cells just receiving irradiation. This breakup of DNA in rad18-2 cells is not degradation due to cell death since wild type cells irradiated to similar low survival levels do not show this breakup of DNA with 8 h incubation. The breakup of DNA in rad18-2 cells is not due to replication gaps being formed by synthesis on a damaged template since treatment of rad18-2 a mating type cells with alpha factor, to prevent initiation of DNA synthesis, does not prevent breakup of the DNA.

Repair of interstrand cross-links in DNA of Saccharomyces cerevisiae requires two systems for DNA repair: the RAD3 system and the RAD51 system.

We have studied the role of the excision-repair system and the recombination-repair system in the removal of cross-links and monoadducts caused by furocoumarins plus 360 nm radiation in yeast DNA by neutral and alkaline sucrose gradients and by a fluorometric procedure which detects cross-linked DNA molecules. We found that the excision-repair system, represented by the rad3 mutations, is required both for the removal of monoadducts, causing single-strand break formation, and for the removal of cross-links, causing double-strand break formation. The recombination-repair system, represented by the rad51 mutation, is necessary for double-strand break repair following cross-link removal, but it has no role in the repair of monoadducts. It can be concluded, that at least some of the same enzymes are used in yeast for both the excision of pyrimidine dimers and the excision of cross-links or monoadducts caused by furocoumarins plus light. The RAD3 and RAD51 repair systems, which act independently in the repair of UV-induced lesions, are part of a single system for the repair of cross-links.