araC-treated Cells Research Articles

Abstract 1185: Translational study of the enhancement of cytarabine toxicity to AML blasts by a differentiation agent combination

Abstract Cytarabine (AraC) is an important component of AML therapy, but like other DNA damaging therapeutic agents it is rarely curative. Exploratory studies suggest that therapy outcomes may be improved by combining AraC with other compounds. In this context, we have recently reported that the addition of a vitamin D-based differentiating agent combination following AraC damage to AML cells in established culture, increases the cell kill. Here we present data obtained with AML blasts cells freshly obtained from patients with this disease which demonstrate that HL60 and U937 cell lines provide a good model to supplement studies of the mechanisms underlying this potential therapeutic regimen. Importantly, in contrast to malignant cells, mononuclear cells isolated from normal human bone marrow do not show the enhancement of AraC-induced cell death by combinations of differentiation agents. The cells were exposed to AraC at concentrations which produced approximately 30-40% cell kill, followed by a combination of 100 nM 1alfa-hydroxyvitamin D2 (D2) and 10 uM carnosic acid (CA), which together serve as a powerful differentiating agent combination (D2/CA) for AML cells, but are minimally toxic alone. Increased cell death, measured by Annexin V/Propidium Iodide, compared to AraC alone, was selective for malignant cells, occurred by both apoptosis and necrosis, and was associated with increased levels of vitamin D receptor (VDR), DNA damage, and higher levels of DNA damage response marker P-H2AX. The knock-down of VDR with siRNA markedly reduced the potentiating effect of D2/CA on the AraC- induced cell death and the associated cellular changes. Since several caspases showed increased expression, and a caspase 3-specific inhibitor markedly reduced the enhancement of cell death, we measured the levels of the pro-apoptotic member of the Bcl family, Bim, which prominently increased following the addition of D2/CA to cells previously exposed to AraC. The increased Bim expression occurred at both mRNA and protein levels. Consistent with a role in the enhancement of cell death in this system, knock down of Bim by siRNA abrogated the effect of adding D2/CA to AraC-treated cells. Thus, our data indicate that in AML cells with pre-existing DNA damage activated VDR can interact with apoptotic pathways, and rather than promoting differentiation, leads to increased leukemia cell kill. (Supported by grant R01CA044722-25 from the National Cancer Institute, NIH, and grant 10A049 from American Institute for Cancer Research, both to GPS; JSH received support from the Nellie B. Smith Endowment at the University of Missouri and the Ellis Fischel Cancer Research Fund at the University of Missouri). Citation Format: Xuening Wang, Jonathan Harrison, George P. Studzinski. Translational study of the enhancement of cytarabine toxicity to AML blasts by a differentiation agent combination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1185.

Is RNA Binding in the Rossmann Fold Essential for GAPDH Intranuclear Functions?

In addition to its prominent enzymatic activity, GAPDH is an enigmatic component of multiple unrelated biochemical entities. In this study, we explored a series of mutated GAPDH and fusion EGFP-GAPDH polypeptides and compared nuclear accumulation, intranuclear mobility and RNA binding properties of the wild type and variant GAPDH proteins. Our results revealed that RNA binding to T99I-mutated GAPDH with non-functional NAD+ binding center occurred outside the Rossmann fold. At the cellular level, wild type and mutated EGFP-GAPDH demonstrated distinct intranuclear localization in unstressed cells versus cells exposed to genotoxic stress. Wild type EGFP-GAPDH protein localized in the cytoplasm of untreated cells and accumulated in the nucleus following araC treatment (11.8±2.72% vs. 27.4±4.28% nuclear EGFP-GAPDH, % of total EGFP-GAPDH, p = 0.0007). Mutated T99I EGFP-GAPDH accumulated at high level in the nuclei of untreated and araC-treated cells (34.3±8.49% vs. 41.3±16.0% nuclear EGFP-GAPDH, % of total EGFP-GAPDH, p = 0.21). Mutated T99I EGFP-GAPDH lost its ability to form tight interactions with intranuclear macromolecules. After araC treatment, immobile fraction (1-Mf) of wild type EGFP-GAPDH in the nuclei of SW48-297 cells was three times higher (0.75±0.127 vs. 0.26±0.133%, p<0.0001), recovery half-time was three times higher (0.84±0.075 vs. 0.3±0.085 s, p<0.0001) and diffusion coefficient D was five times lower (4.6±0.85 vs. 23.3±13.79 μm2/s, p = 0.0001) compared to cells expressing T99I variant. Our results suggest that the switch between RNA and NAD+ binding to GAPDH could be a regulatory mechanism governing participation of GAPDH in NAD+-dependent complexes and could serve a depot to supply diverse biochemical processes with NAD+.

Triptolide cooperates with chemotherapy to induce apoptosis in acute myeloid leukemia cells

Triptolide has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro. The THP1 cell line and primary acute myeloid leukemia (AML) cells were cultured with triptolide alone or in association with AraC or idarubicin in increasing concentrations. Apoptosis was measured by flow cytometry using DiOC6(3) for the cell line and fluorescein isothiocyanateAnnexin-V and CD45 labeling for fresh blast cells. Protein expression was measured by Western blot. Cell cycle distribution of apoptotic cells was measured by flow cytometry. A synergistic effect was observed when triptolide was added to idarubicin or to AraC to induce apoptosis of THP-1 leukemic cells. The triptolide/AraC association was also investigated in vitro on primary blast cells from 25 AML patients. This combination induced significantly higher percentages of apoptosis vs treatment with each drug separately (p<0.005). The IkappaB and X-linked inhibitor of apoptosis protein contents, which were altered by triptolide in idarubicin-treated cells, were not modified in AraC-treated cells. The association of AraC with triptolide increased the number of cells blocked in the S phase and most underwent apoptosis. These results suggest that, by modifying the cell cycle kinetics, AraC sensitizes AML cells to apoptosis induced by low concentration triptolide. The in vitro proapoptotic effect of triptolide associated with the antiproliferative activity of AraC warrants further clinical investigation for treatment of AML patients, especially elderly patients for whom low-dose AraC treatment could be improved by the addition of triptolide.

GH prevents apoptosis in cardiomyocytes cultured in vitro through a calcineurin-dependent mechanism.

The use of GH to treat heart failure has received considerable attention in recent years. Although the mechanisms of its beneficial effects are unknown, it has been implicated in the regulation of apoptosis in several cell types, and cardiomyocyte apoptosis is known to occur in heart failure. We therefore decided to investigate whether GH protects cardiomyocytes from apoptosis. Preliminary experiments confirmed the expression of the GH receptor (GHR) gene in primary cultures of neonatal rat cardiomyocytes (PC), the specific binding of GH by HL-1 cardiomyocytes, and the GH-induced activation of GHR and its classical downstream effectors in the latter. That GH prevented the apoptosis of PC cells deprived of serum for 48 h was shown by DNA electrophoresis and by Hoechst staining assays in which GH reduced the percentage of cells undergoing apoptosis. Similarly, the TUNEL-evaluated pro-apoptotic effect of cytosine arabinoside (AraC) on HL-1 cells was almost totally prevented by pre-treatment with GH. Fluorescence-activated cell sorter (FACS) analysis showed apoptosis in 9.7% of HL-1 cells growing in normal medium, 21.1% of those treated with AraC and 13.9% of those treated with AraC+GH, and that GH increased the percentage of AraC-treated cells in the S/G(2)/M phase from 36.9% to 52.8%. GH did not modify IGF-I mRNA levels or IGF-I secretion in HL-1 cells treated with AraC, and the protection afforded by GH against AraC-induced apoptosis in HL-1 cells was not affected by the presence of anti-IGF-I antibodies, but was largely abolished by the calcineurin-inhibiting combination cyclosporin+FK506. GH also reduced AraC-induced phosphorylation of mitogen-activated protein kinase p38 (MAPK p38) in HL-1 cells. In summary, GH protects PC and HL-1 cells from apoptosis. This effect is not mediated by IGF-I and may involve MAPK p38 as well as calcineurin.

Imbalanced DNA synthesis induced by cytosine arabinoside and fludarabine in human leukemia cells

Previous studies have demonstrated that cytosine arabinoside (araC) induces an accumulation of Okazaki fragments, while fludarabine (FaraA) inhibits Okazaki fragment synthesis. We extended these observations in the present study to provide insights into various mechanisms by which these anticancer drugs affect DNA replication and induce genomic instability in human CEM leukemia cells. Neither araC nor FaraA induced a detectable amount of re-replicated DNA in S-phase cells, which indicated that drug-induced alterations in Okazaki fragment synthesis were not accompanied by DNA re-replication. Synthesis on both leading and lagging DNA strands within the c- myc locus was measured in cells incubated with equitoxic concentrations of araC or FaraA. In araC-treated cells, nascent DNA from the lagging strand was enriched about 5-fold compared with the leading strand. In contrast, FaraA did not induce any replication imbalance. AraC- and FaraA induced changes in the frequency of N-(phosphonacetyl)-l-aspartate (PALA) resistance and the extent of CAD gene amplification were monitored as markers of drug-induced genomic instability. At concentrations that reduced cloning efficiency by 50% ( ic 50), araC increased the frequency of PALA resistance about 4-fold, while FaraA did not have a significant effect on the frequency of PALA resistance. Pretreatment with araC also increased the extent of CAD gene amplification. We propose that the imbalanced DNA synthesis induced by araC leads to the accumulation of Okazaki fragments on the lagging arms and single-stranded DNA regions on the leading arms of replication forks. The formation of these abnormal replication structures was associated with the generation of genomic instability.

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis.

Treatment with cytosine beta-D-arabinoside (AraC; 300 microM) induced a time-dependent accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase-3. Moreover, Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-fmk), a CPP32-selective antagonist, dose-dependently suppressed AraC-induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil-DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC-treated cells. The more alkaline isoforms, 1-3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4-6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h; a 4-h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside-induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC-induced neuronal death.

Cytogenetic response of normal human and ataxia telangiectasia G 2 cells exposed to X-rays and ara C

The kinetics of rejoining of chromatid deletions and the formation of exchanges has been studied in X-irradiated normal and ataxia telangiectasia (AT) fibroblasts treated in the presence or absence of the nucleoside analogue 1-β- d-arabinofuranosylcytosine (ara C). Ara C is a powerful inhibitor of DNA synthesis as well as an inhibitor of DNA double-strand break repair. Treatment with ara C was found to increase the frequency of X-ray-induced chromatid deletions in both lines with increasing incubation time while deletions were found to rejoin with first-order kinetics and a t 1 2 of 2.4–3.1 h in both cell lines. The increase in deletions in the presence of ara C is thought to be due to an interaction of ara C-induced lesions (as yet unidentified) with lesions induced by X-rays, leading to additional chromatid breaks. These results are in contrast to those previously obtained with the same lines treated with X-rays and 9-β- d-arabinofuranosyladenine (ara A). In this case the frequency of deletions in X-irradiated cells remained constant in both lines in the presence of ara A. We therefore propose that there is a major difference in the mode of action of ara C and ara A on X-ray-induced DNA damage in G 2 cells. Exchanges were formed in X-irradiated cells in the presence and absence of ara C in both lines and the frequency increased with post-irradiation incubation time. A higher frequency was formed in ara C-treated cells than in cells given X-rays alone, but the enhancement by ara C was less than that previously found in cells treated with ara A.

The presence of non-neuronal cells influences somatostatin release from cultured cerebral cortical cells

We examined the effect of non-neuronal cells on somatostatin release from cultured cerebral cortical cells. Three culture models were used: (1) neuron-enriched cultures obtained from cortex of 17-day-old rat embryos and exposed to 10 μM cytosine arabinoside (Ara C) for 48 h between days 3 and 5 after plating; (2) whole cell cultures obtained by using the same protocol but untreated with Ara C; (3) glial primary cultures obtained from newborn rats. We studied: (i) the cellular composition of the cultures by using two astroglial markers: vimentin and glial fibrillary acidic protein (GFAP); (ii) the spontaneous and forskolin-stimulated somatostatin release. In 8-day-old cultures morphological data revealed that Ara C treatment reduced glial cells to 6%. At 7 and 10 days of culture somatostatin spontaneously released from Ara C-treated cells was higher than that measured from untreated cells. On the 17th day of culture, neuron-enriched cultures contained a lower amount of somatostatin than whole cell cultures. Forskolin elicited a dose-dependent release of somatostatin from whole cell cultures, but had no effect on neuron-enriched cultures. Astroglial released media (ARM) from glial primary cultures exposed to forskolin for 20 min induced somatostatin release from neuron-enriched cultures. HPLC analysis of endogenous amino acids of ARM showed that glutamate, glutamine, glycine and alanine were significantly increased after forskolin stimulation. Our results suggest a functional interaction between glial cells and neurons secreting somatostatin.

Activation of a well behaved cell cycle in araC-treated V79 cells by caffeine

0.3–1.0 σmM araC (cytosine arabinoside) treatment of V79 cells produced inhibition of multiplication of cells which was accompanied by a large increase of cell size. In presence of 1–2 mM caffeine the inhibition of cell proliferation due to araC treatment was substantially reduced and cell-size increase was prevented; caffeine did not influence the uptake of araC by V79 cells. Flow microfluorometric analysis showed that caffeine induced a wave of cell cycle progression in 0.3 μM araC-treated cells. The cell cycle activated by caffeine in 0.3 μM araC-treated cells was largely well behaved; this was indicated by the fact that (1) prior to cell division cells achieved a tetraploid DNA content and (2) following cell division they had diploid DNA content as a result of which DNA homeostasis was maintained. At 1.0 μM araC concentration, however, extreme micronucleation was observed which gave rise to a substantial fraction of micronuclei with < G1 DNA content.

The effect of cytosine arabinoside on the frequency of single-strand breaks in DNA of mammalian cells following irradiation or chemical treatment

1-β- d-Arabinofuranosyl cytosine (araC), a pyrimidine nucleoside analog used in the treatment of malignant tumors [1,2], inhibits ultraviolet repair of DNA in a reversible manner. The inhibition occurs during the resynthesis-ligation step and is apparent at all sites undergoing repair. By use of araC it was possible to substantiate the reported observation that the initial velocities of ultraviolet repair are dose dependent and that hamster and human cells are more efficient that mouse cells in excising DNA damage after fluences of less than 50 J/m 2. araC does not strongly inhibit γ-ray-induced repair, although alkali-labile sites are removed more slowly in araC-treated cells. Repair of damage to DNA by N- methyl-N- nitrosoguanidine , mitomycin C, 4-nitroquinoline oxide and 8-hydroxyquinoline is strongly inhibited by araC.