There are numerous Publication Only "sessions". Some follow. [I'm getting tired.<G>]
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[4318] Arsenic Trioxide Induced p53-Dependant Apoptotic Signals in a Temperature Sensitive (Ts) p53 Mutant, BRK Cell Line. Session Type: Publication Only
Cagla Akay, Yair Gazitt. Medicine, University of Texas Health Science Center, San Antonio, TX, USA
Mutations in the p53 gene are the most common genetic abnormality in human cancers and are the cause for drug resistance. Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia (APL) and is being tested in phase II studies in various types of cancers. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53 whereas in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. To further establish the differential effect of ATO in relation to the p53 status we studied the effect of temperature shift in temperature sensitive (Ts)-p53 expressing baby rat kidney (BRK) cells. We studied by Western immunoblotting the activation of the intrinsic and the extrinsic apoptotic pathways in ATO-induced apoptosis of BRK cells cultured at 32°C (w.t. p53 phenotype) and 37°C (mutant p53 phenotype). As expected, at 32°C, we observed a G1 arrest through activation of p21. We also observed depolarization of mitochondrial membrane; the release of cytochrome C and activation of caspase-9 and apoptosis as measured by Annexin V. We also observed release of SMAC at 32°C. In contrast, at 37°C we observed a G2/M arrest with no activation of p21 with activation of the extrinsic apoptotic pathway through early induction of TRAIL and TRAIL receptor- R2, activation of caspase-8, activation of BID, degradation of FLIP, rapid depolarization of mitochondrial membrane and release of AIF from mitochondria to the cytosol. We also demonstrate by flow cytometry and confocal imaging translocation of AIF to the nucleus in ATO-induced apoptosis at 37°C but not at 32°C. These results further substantiate our p53 model (Akay and Gazitt, Cell Cycle 2:258-268, 2003).
Abstract #4318 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Apoptosis|Arsenic trioxide|p53
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[4352] Effects of Arsenic Trioxide on Protein Kinase C and Cytosolic Calcium in Ventriclar Myocardium of Guinea Pig. Session Type: Publication Only
Jin Zhou, Ran Meng, Lu Meng, Baofeng Yang (Intr. by Jialan Shi). Hematology, The First Hospital of Harbin Medical University, Harbin, Heilongjiang, China; Neurology, The First Hospital of Harbin Medical University, Harbin, Heilongjiang, China; Biochemistry & Molecular Biology, University of Georgia, Athens GA, Athens, GA, USA; Phamacology, The First Hospital of Harbin Medical University, Harbin, Heilongjiang, China
AIM To clarify the mechanisms of the cardiac side effects of arsenic trioxide (As2O3) in leukemia treatment, the effects of As2O3 on protein kinase C(PKC) and cytosolic calcium ([Ca2+]i) in ventriclar myocardium of guinea pig were investigated.
METHODS Fluo-3/AM fluorescent probe tagged [Ca2+]i of guinea pig’s ventriclar myocardium, detected the [Ca2+]i, by laser confocal microscopy before and after exposed to As2O3. Phosphorus radioisotope assay was used to detect the activity of PKC on cell membrane or in cytoplasm. DNA ladders of ventriclar myocardium in Tyrode’s solution after exposed to As2O3 were evaluated.
RESULTS The cytosolic [Ca2+]i did not change when incubated guinea pig’s ventriclar myocardium in Tyrode’s solution with 0.5µmol·L-1As2O3, after incubated in2µmol·L-1As2O3 for 6min, [Ca2+]i began to increase and was continuous increasing for about 9min. After exposed to 10µmol ·L-1As2O3 for less than 3 min, the [Ca2+]i kept on increasing till the end (about 27min) of the examination and could not recover. The increasing of [Ca2+]i could be partly blocked by calcium channel blocking agents—verapamil and nifedipin. The PKC of myocardial cells began to rise after incubated in an As2O3 changing concentration culture media for 3 hours , which initial As2O3 was 10µmol·L-1, and the DNA ladders appeared after incubated in it for10 hours. No DNA ladders appeared when incubated in an As2O3 culture media in which the initial As2O3 concentration was under 5µmol·L-1.
CONCLUSIONS As2O3 can active PKC, increase cytosolic [Ca2+]i and promote apoptosis of myocardial cells. The promoting apoptosis action of As2O3 is closely related to the lasting activation of PKC. The initial action target of As2O3 is cell membrane.
Abstract #4352 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Acute myeloid leukemia|APL|As2O3
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[4361] Arsenic Trioxide Induces Apoptosis in Molt-4 Cell Lines: Caspase 8-Dependent and Caspase 3-Independent. Session Type: Publication Only
Wenbin Qian, Wanmao Ni, Junqing Liu (Intr. by Jiong Hu). Hematology, The First Hospital affiliated to Medical College,Zhejiang University, Hangzhou, Zhejiang, China
Arsenic Trioxide (As2O3 ) has been used successfully in the treatment of patients with relapsed or refractory acute promyelocytic leukemia (APL) without severe marrow suppression. Currently, the action of As2O3 on many other hematopoietic malignancies is under investigation. Much evidence has shown that caspase-3 plays essential executing role in apoptosis of many leukemia cell lines. But, the exact mechanism of As2O3-induced apoptosis in Molt-4 cell line which is originated from acute lymphoblastic leukemia is not well understood. Here, we investigate the action of As2O3 on Molt-4 cells and involved mechanism. Significant dose- and time-dependent inhibition of cell growth was observed by MTT assay. Following the treatment of As2O3 for 72 h, As2O3 at 4 µM exhibited 50% inhibition of growth in Molt-4 cells. The effect of As2O3 on the cell cycle was determined in Molt-4 cells by FACS analysis. DNA flow cytometric analysis with three independent experiments indicated that As2O3 induced a G1 and a G2-M phase arrest in Molt-4 cells following 6µM of exposure. Similar results were observed in Molt-4 cells following 2µM and 4µM exposure. These results indicated that As2O3 inhibited the cellular proliferation of Molt-4 cells via a G1 and a G2-M phase arrest of the cell cycle. To confirm and evaluate the induction of apoptosis, we performed the staining of cells with annexin V and PI. As with the percentages of sub-G1 group by FACS analysis, the proportion of apoptotic cells was increased in a dose-and -time dependent manner. Taken together, these results indicate that induction of apoptosis can be another mechanism of the antiproliferative effect of As2O3 besides G1 and G2-M phase arrests of the cell cycle in Molt-4 cells. We subsequently studied the activation of initiator caspase-8 and executioner caspase-3 in Molt-4 cells by Western blotting. Molt-4 cells that had undergone apoptosis on culturing with As2O3 displayed the initial activation of caspase-8 with the appearance of the large cleavage fragment of 43 to 41 kd. Despite the higher basal level of procaspase-3 expression in the Molt-4 cells prior to As2O3 treatment, we were unable to detect cleaved, activated caspase-3 following As2O3 treatment. Next, we checked whether inhibition of caspases-3 could abrogate the proapoptotic effects of As2O3. For this purpose the caspase-3 inhibitor, z-DEVD-fmk, was used. The results shown that addition of z-DEVD-fmk did not rescue Molt-4 cells from apoptosis induced by As2O3. These results clearly differ from other observations made with other leukemia cells and might explain, at least in part, that As2O3 induces apoptosis in Molt-4 cells is caspase 8-Dependent and caspase 3-Independent.
Abstract #4361 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Arsenic trioxide|Apoptosis|Caspase
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[4450] Effect of Administration Styles of Arsenic Trioxide on the Intracellular Arsenic Concentration and the Efficiencies of Differentiation and Apoptosis. Session Type: Publication Only
Jin Zhou, Ran Meng, Baofeng Yang (Intr. by Jialan Shi). Hematology, The First Hospital of Harbin Medical University, Harbin, Heilongjiang, China; Neurology, The First Hospital of Harbin Medical University, Harbin, Heilongjiang, China; Phamacalogy, The First Hospital of Harbin Medical University, Harbin, Heilongjiang, China
Objective: Study on the effects of As2O3 on intracellular arsenic concentration, differentiation and apoptosis rates and sensitivities by different administration styles.
Methods: Evaluated the intracellular arsenic concentrations ,the apoptosis rates, and the ratios of differentiation phenotypes on cell surface CD33 (–) /CD11b (+), when incubated cells with As2O3 in different culture media in vitro and treated leukaemia patients with As2O3 in different administration styles in vivo.
Results: When incubated cells in a constant arsenic concentration culture medium for 24 hours, the intracellular arsenic concentrations were (33.4±0.88) µg/L in NB4, (18.6±1.12) µg/L in K562, and (28.8±0.64) µg/L in APL; the apoptosis rates were NB4 56.6%, K562 27.6%, APL 52.2%; the ratios of differentiation phenotype CD33(-) / CD11b(+) were: NB4 (19.5±0.5)%, K562 (24.5±0.6)%, APL (18.5±0.6)% , whereas , when incubated cells above in a changing arsenic concentrations culture medium for 24hours, which initial arsenic level was 5µmol/L, the intracellular arsenic concentrations were : NB4(17.6±0.88) µg/L, K562(9.2±0.64)µg/L, APL(15.2±1.04)µg/L, the apoptosis rates were: NB4 23.2%, K562 11.0%, APL 21.0%; the ratios of differentiation phenotype CD33 (–) /CD11b (+) were : NB4 (51.5±0.4)%, K562 (57.5±0.8)%, APL (46.5±0.5)%. 24hours after one time continuous and slowing intravenous As2O3 infusion , the intracellular arsenic concentrations were APL (26.6±2.5) µg/L,AML-M2 (15.5±3.1) µg/L, CML (18.5±2.3) µg/L; the apoptosis rates were APL 28.5%, AML-M2 9.5%, CML 12.5%; the ratios of differentiation phenotype CD33 (–)/CD11b (+) were : APL (29.5±0.5)%, AML-M2 (28.5±0.6) %, CML (23.5±0.5)%. Whereas, when 24hours after treated with one time general speed intravenous As2O3, the intracellular arsenic concentrations were : APL(12.3±2.1) µg/L, AML-M2 (5.5±2.3) µg/L , CML (8.5±2.7) µg/L ; the apoptosis rates were : APL 13.2%, AML-M2 3.9%, CML4.2%; the ratios of differentiation phenotype CD33 (–) /CD11b (+) were: APL (53.6±0.8) %, AML-M2 (48.5±0.9) % , CML (67.5±0.8) %.
Conclusions: The administration styles affect the intracellular arsenic concentrations, the differentiation rates and the ratios of apoptosis of leukaemia cells. Treated with the continuous and slowing intravenous As2O3 infusion can obtain a higher intracellular arsenic concentration, a higher efficiency of apoptosis and a lower differentiation ratio than that in general speed intravenous As2O3 infusion in clinical dosage. The continuous and slowing intravenous As2O3 infusion can promote apoptosis and inhibit from differentiation, so this As2O3 medication can relieve leukocytosis and elevate therapeutic effects.
Abstract #4450 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Acute myeloid leukemia|APL|As2O3
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[4459] Effects of Arsenic Trioxide on Megakaryocytic Cell Lines: Apoptosis, Cell Cycle Dysfunction and Signaling Pathways. Session Type: Publication Only
Hubert K.B. Lam, Karen K.H. Li, Ki Wai Chik, Mo Yang, Carmen K.Y. Chuen, Venus C.T. Liu, Chi Kong Li, Matthew M.K. Shing, Patrick M.P. Yuen. Department of Paediatrics, The Chinese University of Hong Kong, Shatin, NT, Hong Kong
Despite progress made in the elucidation of the actions of arsenic trioxide (ATO) in acute promyelocytic leukemia, the molecular mechanisms leading to apoptosis in other malignancies remain unclear. In particular, the effects of ATO on the megakaryocytic (MK) lineage have not been well characterized. In this study, we focused on two MK cell lines CHRF-288-11 (CHRF) and MEG-01, which were derived from an infant and adult acute megakaryocytic leukemia (AMKL), respectively. Our data showed that these cells underwent apoptosis within 24 - 48 h post-ATO (6 µM) treatment, as demonstrated by the Annexin V assay (Table 1). By flow cytometry, significant activation of caspase-3 was detected in the MK cells at 24 h, and was preceded by the loss of mitochondrial membrane potential (8 h) as determined by the fluorescent dye JC-1. Western blotting experiments showed that ATO induced Bax expression and down-regulated Bcl-2, which led to an increase in Bax/Bcl-2 ratio. ATO exerted immediate and significant interference on the cell cycle by delaying S-phase progression and the subsequent accumulation of cells in the G2/M phase (43.2% vs 13.6%, p < 0.01). By multivariate analysis (BrdU and 7-AAD), active caspase-3 was detected in all phases of the cell cycle. The responses of CHRF and MEG-01 cells to ATO were similar, except that the latter appeared more resistant, in terms of the dosage of ATO and the slight delayed onset of apoptosis. We screened the expression levels of 96 genes involved in apoptosis using the GEArray Q Series Human Apoptosis Gene Array at 0, 4, 8 and 16 h (each n = 2) post-ATO treatment. We identified the up-regulation of mRNA of two extrinsic components of apoptosis. Fas was progressively increased in both cell lines (up to 6.14-fold) and caspase-8 was elevated in MEG-01 (3.58-fold). The protein expressions of Fas and activated caspase-8 were demonstrated in both cell lines by flow cytometry. Increased mRNA expressions of caspase-1 (2.30-fold) and CD137 (2.33-fold) were also noted, but their significance in apoptosis of our system remained to be investigated. To demonstrate the direct effect of ATO on gene expressions in AMKL cells, a more comprehensive microarray (Human 19K Array, Ontario Cancer Institute Microarray Centre) was used. Treatment with ATO for 4 h (n = 3) prompted an elevation in the mRNA levels of stress-associated proteins, such as metallothioneins (MT1G: 6.31-fold; MT2A: 3.64-fold), Hsp72 (5.81-fold), Hsp73 (3.77-fold), Hsp90 (2.11-fold), ferritin (2.02-fold) and ubiquitin (2.76-fold). Interestingly, WT1, a cell cycle regulatory gene elevated in many types of leukemia, was induced by ATO (2.44-fold). In conclusion, our results suggested that apoptosis in AMKL cells mediated by ATO involved a switch from pro-survival in the early phase to the activation of multiple death machineries, consisting of the intrinsic (mitochondrial, Bax, Bcl-2) and the extrinsic (Fas, caspase-8) compartments.
Table 1: Signals regulated by ATO in CHRF cells 0 h 24 h 48 h
Annexin V +/PI - (%) 4.56±0.28 8.28±0.53* 9.83±0.73*
Active caspase-3 (%) 2.28±0.13 4.58±0.87* 14.7±1.16*
JC-1 greenhi/redlo (%) 4.18±0.52 8.05±0.60* 20.76±8.69*
Bax/Bcl-2 (Fold)# 0.63±0.08 2.65±0.68 -
Fas (Fold) 1 1.73±0.17* 1.96±0.20*
CD137 (Fold) 1 1.55±0.08* 1.76±0.03*
Mean ± SEM; * p < 0.05 compared to 0 h; # n = 2, others n = 3-5.
Abstract #4459 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Arsenic trioxide|Signaling|Acute myeloid leukemia
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