Review of clinical and haematological response to low-dose cytosine arabinoside in acute myeloid leukaemia

15 patients with acute myeloid leukaemia (AML) were treated with low-dose cytosine arabinoside (LD ARA-C). 2 patients had complete remissions, which lasted for 8 and 3 months, and 5 patients had a partial remission. 46% of the patients thus responded to LD ARA-C. This included 1 responding patient who had not previously responded to therapy with 6-mercaptopurine, thioguanine, or vinblastine. The 2 patients with complete remission did not show LD ARA-C-induced hypoplasia of bone marrow, although 1 had hypoplastic AML before therapy. Leukaemic cells from 1 patient showed in vivo maturation from M1 to M3 after LD ARA-C treatment. The present results, together with the published data, indicate that: a. LD ARA-C treatment, although it may have some toxic effects, is an effective treatment for some patients with AML, especially those with hypoplastic AML; b. Response to LD ARA-C can be obtained after one or several courses of treatment; c. LD ARA-C-induced remissions are sometimes obtained even in patients who fail in more conventional treatments; d. LD ARA-C-induced remissions can be achieved without bone marrow hypoplasia, and induction of hypoplasia by itself does not always result in complete remission; e. LD ARA-C can induce in vivo maturation of leukaemic cells. It is suggested that induction of remission in AML patients by LD ARA-C may result from either differentiation of leukaemic blast cells, cytotoxicity to leukaemic blasts, or both mechanisms acting together.     

Lysosomal destabilization in p53-induced apoptosis

The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37 degrees C to 32 degrees C. We have now found that these cells showed an early lysosomal rupture after transfer to 32 degrees C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca(2+)-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders.     

Oxidative stress mediates impairment of muscle function in transgenic mice with elevated level of wild-type Cu/Zn superoxide dismutase

Cases of familial amyotrophic lateral sclerosis (fALS; a neurodegenerative disorder) have been reported in which the gene for Cu/Zn superoxide dismutase (CuZnSOD) was mutated. Several studies with the fALS mutant CuZnSOD in transgenic mice and cells showed that the fALS mutations act through an as yet undefined dominant gain-of-function mechanism. Wild-type CuZnSOD catalyzes the dismutation of superoxide (O₂) but also produces hydroxyl radicals (^•OH) with H₂O₂ as substrate. Two laboratories have recently demonstrated that the ^•OH production ability was preferentially enhanced by the fALS mutant CuZnSOD, suggesting that this might be the function gained in fALS. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of wild-type CuZnSOD was also associated with increased ^•OH production and impaired muscle function. Enhanced formation of ^•OH was detected, by spin trapping, in brain and muscle extracts of the Tg-CuZnSOD mice. Three independently derived Tg-CuZnSOD lines showed muscle abnormalities, reflected by altered electromyography (EMG) and diminished performance in the rope grip test. After treatment with paraquat (PQ), a widely used herbicide and O₂-generating compound, muscle disability significantly deteriorated in Tg-CuZnSOD mice but not in control mice. The results indicate that elevated levels of CuZnSOD cause indigenous long-term oxidative stress leading to impairment of muscle function. These findings may provide valuable clues about the concurred role of indigenous oxidative stress and exogenous agents in the etiology of sporadic ALS and several other neurodegenerative diseases in which a specific subset of neurons is affected.     

Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis

M1 myeloid leukemic cells overexpressing wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1β-converting enzyme (ICE)-like proteases, resulting in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these proteases and apoptosis were suppressed by the cytokine interleukin 6 or by a combination of the cytokine interferon γ and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were suppressed by some protease inhibitors. In a clone of M1 cells that did not express p53, vincristine or doxorubicin induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by interleukin 6. In another myeloid leukemia (7-M12) doxorubicin also induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by granulocyte–macrophage colony-stimulating factor. The results indicate that (i) overexpression of wild-type p53 by itself or treatment with cytotoxic compounds in wild-type p53-expressing or p53-nonexpressing myeloid leukemic cells is associated with activation of ICE-like proteases; (ii) cytokines exert apoptosis-suppressing functions upstream of protease activation; (iii) the cytotoxic compounds induce additional pathways in apoptosis; and (iv) cytokines can also suppress these other components of the apoptotic machinery.     

Inhibition of p53-induced apoptosis without affecting expression of p53-regulated genes

Using DNA microarray and clustering of expressed genes we have analyzed the mechanism of inhibition of wild-type p53-induced apoptosis by the cytokine interleukin 6 (IL-6) and the calcium mobilizer thapsigargin (TG). Clustering analysis of 1,786 genes, the expression level of which changed after activation of wild-type p53 in the absence or presence of IL-6 or TG, showed that these compounds did not cause a general inhibition of the ability of p53 to up-regulate or down-regulate gene expression. Expression of various p53 targets implicated as mediators of p53-induced apoptosis was also not affected by IL-6 or TG. These compounds thus can bypass the effect of wild-type p53 on gene expression and inhibit apoptosis. IL-6 and TG activated different p53-independent pathways of gene expression that include up-regulation of antiapoptotic genes. IL-6 and TG also activated different differentiation-associated genes. The ability of compounds such as cytokines and calcium mobilizers to inhibit p53-mediated apoptosis without generally inhibiting gene expression regulated by p53 can facilitate tumor development and tumor resistance to radiation and chemotherapy in cells that retain wild-type p53.     

Autologous cell vaccine as a post operative adjuvant treatment for high-risk melanoma patients (AJCC stages III and IV). The new American Joint Committee on Cancer

This study evaluates the overall survival and disease free survival of melanoma patients that were treated with an autologous melanoma cell vaccine, administered as a post-operative adjuvant. Included are 43 patients with totally resected metastatic melanoma (28-AJCC stage III, 15-AJCC stage IV), with a median follow up of 34 months (6-62). The treatment consisted of eight doses of a vaccine made of 10-25x10(6) autologous melanoma cells either released from the surgical specimen or grown in cell cultures. Tumour cells were conjugated with hapten dinitrophenyl, mixed with Bacille Calmette Guérin and irradiated to 110 Gy. Both disease free survival and overall survival were found to be correlated with intensity of evolving delayed type hypersensitivity to subcutaneous injection of unmodified melanoma cells. Patients with a delayed type hypersensitivity reaction of > or =10 mm had a median disease free survival of 17 months (mean 35 months) and a mean overall survival of 63 months (median not reached). In contrast, patients with a negative or weak delayed type hypersensitivity had a median disease free survival of 9 months (relative risk of recurrence=4.5, P=0.001), and a median overall survival of 16 months (relative risk of death=15, P=0.001). Stage III patients with a positive delayed type hypersensitivity reaction had an improved disease free survival of 16 months and a mean overall survival of 38 months, whereas patients with a negative delayed type hypersensitivity had a median disease free survival of 7 months (relative risk=4.5, P=0.02) and a median overall survival of 16 months (relative risk=9.5, P=0.005). The adjuvant administration of autologous melanoma vaccine was associated with improved disease-free and overall survival to selected patients who successfully attained anti-melanoma reactivity as detected by positive delayed type hypersensitivity reactions to unmodified melanoma cells.     

Correlation of toxicity with pharmacokinetics of pegylated liposomal doxorubicin (Doxil) in metastatic breast carcinoma

BACKGROUND: Doxil (ALZA Corp., Mountain View, CA) is a formulation of doxorubicin in polyethylene-glycol coated liposomes with a prolonged circulation time and unique toxicity profile. As yet, the effect of the dose schedule on toxicity and the correlation of toxicity with pharmacokinetics have not been directly addressed. METHODS: The objectives of this study were to examine the toxicity profile and pharmacokinetics of various dose schedules of Doxil in a group of patients with metastatic breast carcinoma (MBC) previously treated with chemotherapy. Forty-five patients received a total of 268 courses of Doxil (median per patient, 5; range, 1-19). Six dose schedules were investigated: 35 mg/m2 every 3 weeks (11 patients), 45 mg/m(2) every 3 weeks (5 patients), 50 mg/m(2) every 4 weeks (5 patients), 60 mg/m(2) every 4 weeks (6 patients), 65 mg/m(2) every 5 weeks (6 patients), and 70 mg/m(2) every 6 weeks (12 patients). Doxil pharmacokinetics was examined in 24 of these patients at the dose levels of 35, 45, 60, and 70 mg/m(2). RESULTS: Stomatitis was dose related, with higher incidence and severity at doses of 60-70 mg/m(2). Skin toxicity in the form of palmar-plantar erythrodysesthesia (PPE) developed usually after two or more courses of treatment and was schedule dependent with shorter dosing intervals leading to increased frequency and severity of skin manifestations. Myelosuppression, mainly as leukopenia/neutropenia, was dose dependent but mild and uncomplicated in most cases. Hair loss was infrequent (< 7%) and always of limited extent. Despite high cumulative doses up to 1500 mg/m(2), cardiac toxicity was observed in only 1 patient who received prior mitoxantrone and mediastinal radiotherapy. Objective responses, improvements, and durable stabilizations were observed in 9, 6, and 14 patients, respectively, indicating significant antitumor activity of Doxil in previously treated MBC patients. Doxil pharmacokinetics was well described by a monoexponential elimination curve with a long T(1/2) (median, 79 hours), a slow clearance (median, 40 mL/hour), and a small volume of distribution (median, 3.9 L). Cmax (peak plasma concentration) and AUC (area under the concentration*time curve) increased linearly with dose with a statistically significant correlation. Correlation analysis of dose and pharmacokinetic parameters with Doxil toxicites revealed that stomatitis grade and leukocyte nadir were correlated strongly with dose and Cmax, and weakly with AUC, whereas PPE grade was correlated significantly with only 1 parameter, T(1/2). CONCLUSIONS: The toxicity of Doxil is dose and schedule dependent and well correlated with pharmacokinetic parameters. Pharmacokinetic guidance of Doxil dosing may be a useful tool.     

Screening for induction of differentiation and toxicity to blast cells by chemotherapeutic compounds in human myeloid leukemia

Bone marrow cells from 9 patients with acute myeloid leukemia and 1 patient with a blast crisis of chronic myeloid leukemia were cultured to determine their ability to be induced to differentiate by different chemotherapeutic compounds. Five of these 10 patients showed differentiation to granulocytic and/or monocytic cells by culture with medium containing the myeloid cell differentiation-inducing protein MGI-2. Actinomycin D induced differentiation in cells from 2 of the patients who did not show differentiation with MGI-2 containing medium. In these 7 patients there was an increase in the ratio of differentiated myeloid cells to blasts. None of these 10 patients showed induction of differentiation by cytosine arabinoside, adriamycin, or daunomycin, but treatment with these compounds showed in some patients an increase in the ratio of differentiated myeloid cells to blasts. The results indicate that this ratio can be increased by differentiation and also in some patients by toxicity to blast cells. With dexamethasone or vinblastine there was no induction of differentiation and no increase in this ratio in any of the 10 patients tested. After in vivo chemotherapy with low dose cytosine arabinoside, cells from one patient showed a similar response in culture to actinomycin D as cells before chemotherapy, whereas in another patient the cells had acquired the ability to respond to actinomycin D. In contrast, after high-dose in vivo chemotherapy with cytosine arabinoside and daunomycin, cells from a third patient seemed to have lost the ability to differentiate in vitro by MGI-2 containing medium or actinomycin D. The results indicate that pre-screening for differentiation-inducing compounds and compounds that show toxicity to blast cells should be useful to select the appropriate compounds to be used for therapy, and that it is advisable to screen the cells both before and after initiation of therapy.     

Regulation of in-vivo differentiation of myeloid leukemic cells by antigen-specific helper T lymphocytes

There are clones of myeloid leukemic cells that can be induced to differentiate in vitro and in vivo by normal macrophage and granulocyte differentiation-inducing protein MGI-2 (= DF). The differentiation of these myeloid leukemic cells in vivo is regulated by a cell mediated immune response which requires T lymphocytes. We now show that differentiation of myeloid cells in vivo can be induced by antigen-specific helper T lymphocytes and that this is associated with the ability of the helper T cells to produce myeloid cell differentiation-inducing protein MGI-2. Antigen specific helper T cells can accumulate at a site that contains the antigen. It is suggested that migration in response to antigen of helper T cells producing differentiation factors may play an important role in inducing in vivo differentiation of leukemic cells.