Leukemogenic Effects of PML-RARá and Mechanisms of ATRA/Arsenic Trioxide in the Treatment of APL

<div><p>(A) In the absence of RA, RARα/RXR heterodimers recruit the transcription corepressor (CoR), which mediates transcriptional silencing by mechanisms that include direct inhibition of the basal transcription machinery and recruitment of chromatin-modifying enzymes. Chromatin modification includes histone deacetylation, which leads to a compact chromatin structure that impairs the access of transcriptional activators. In the presence of physiological concentrations (10⁻⁹–10⁻⁸ M) of RA, the transcription corepressor is released and the coactivator is recruited to the RARα/RXR heterodimer, resulting in histone acetylation (AC) and overcoming of the transcription blockage.</p> <p>(B) PML-RARα fusion protein binds to RARα target genes either on its own or with RXR and then recruits corepressors, leading to transcriptional repression and myeloid differentiation inhibition. PML-RARα oncoprotein sequesters the normal RXR and PML, inhibits the PML/P53 apoptotic pathway, and delocalizes PML and other proteins from the nuclear body. PML-RARα also may affect interferon (IFN) and other signal pathways. Abnormalities in protein tyrosine kinases (e.g., FLT3, c-fms) may collaborate with PML-RARα to cause APL.</p> <p>(C) In the presence of pharmacological doses of ATRA or arsenic trioxide, the PML-RARα fusion is degraded in ways that are dependent on caspases and proteasomes. The degradation of PML-RARα may lead to derepression of transcription suppression and restoration of PML nuclear body structure. The blockade of other signaling pathways is also released, and the anti-apoptotic effect of PML-RARα is lost. ATRA also induces cyclic AMP (cAMP), which reverses the silencing of RXR, induces the expression of RA-induced genes and cyclooxygenase 1 (Cox 1), inhibits angiogenesis, and downregulates tissue factor. Subsequently, ATRA induces terminal cell differentiation, while arsenic trioxide induces partial differentiation and/or apoptosis of APL cells. The effects of ATRA and arsenic trioxide are indicated with red and blue arrows, respectively. AF2, the ligand-dependent transcriptional activation domain contained within the C-terminal E domain of RARα; D522, aspartate at residue 522; K160, lysine at residue 160; K490, lysine at residue 490; RARE, retinoic acid response element; SUG-1, a component of proteasome 19S complex that can bind with the activated AF2 domain of RARα.</p></div

The Three Features of APL

<p>The three features of APL are (A) accumulation of abnormal promyelocytes, (B) fibrinogenopenia and disseminated intravascular coagulation, and (C) the chromosomal translocation t(15;17)(q22;q21), the resultant fusion transcripts, and variants.</p

GSTT1 Deletion Is Related to Polycyclic Aromatic Hydrocarbons-Induced DNA Damage and Lymphoma Progression

<div><p>The interrelationship between genetic susceptibility and carcinogenic exposure is important in cancer development. Polymorphisms in detoxification enzymes of the glutathione-S-transferases (GST) family are associated with an increased incidence of lymphoma. Here we investigated the molecular connection of the genetic polymorphism of <i>GSTT1</i> to the response of lymphocytes to polycyclic aromatic hydrocarbons (PAH). In neoplastic situation, <i>GSTT1</i> deletions were more frequently observed in lymphoma patients (54.9%) than in normal controls (42.0%, P = 0.009), resulting in an increased risk for lymphoma in individuals with <i>GSTT1</i>-null genotype (Odds ratio = 1.698, 95% confidence interval = 1.145–2.518). <i>GSTT1</i> gene and protein expression were accordingly decreased in <i>GSTT1</i>-deleting patients, consistent with activated profile of cell cycle regulation genes. Mimicking environmental exposure using long-term repeat culture with low-dose PAH metabolite Hydroquinone, malignant B- and T-lymphocytes presented increased DNA damage, pCHK1/MYC expression and cell proliferation, which were counteracted by ectopic expression of <i>GSTT1</i>. Moreover, <i>GSTT1</i> expression retarded xenograft tumor formation of Hydroquinone-treated lymphoma cells in nude mice. In non-neoplastic situation, when zebrafish was exposed to PAH Benzo(a)pyrene, molecular silencing of <i>gstt1</i> enhanced the proliferation of normal lymphocytes and upregulated <i>myca</i> expression. Collectively, these findings suggested that <i>GSTT1</i> deletion is related to genetic predisposition to lymphoma, particularly interacting with environmental pollutants containing PAH.</p></div

<i>GSTT1</i>-null genotype is frequently observed in patients with lymphoma.

<p>A: <i>GSTT1</i> deletion detected by copy number variation (CNV) analysis (upper panel) and validated by semi-quantitative PCR (lower panel) in diffuse large B-cell lymphoma (DLBCL, 15/25, 60.0%), T-cell lymphoma (TCL, 7/20, 35.0%) and reactive hyperplasia (1/8, 12.5%). B: SNP data illustrating the distribution of B allele frequencies (BAF) and Log R Ratio (LRR) values across the region of chromosome 22 (22,676,385 bp to 22,717,669 bp) in the <i>GSTT1</i>-deleting cases and normal controls. C: Semi-quantitative PCR validation of <i>GSTT1</i> deletion in expanded sample-set of DLBCL (57/103, 55.3%), TCL (55/101, 54.5%) and normal controls (86/205, 42.0%). *P<0.05 comparing with normal controls.</p

<i>GSTT1</i> expression protects lymphoma cells from PAH-induced DNA damage.

<p>A: <i>GSTT1</i> gene expression assessed by semi-quantitative PCR in Namalwa and Jurkat cells transfected with <i>GSTT1</i> (GSTT1) and the negative control vector (FU). B: Images represent results from three independent experiments. GSTT1 protein expression detected by immunohistochemistry assay. C: DNA damage measured by alkaline and modified comet assay in Namalwa and Jurkat cells treated with Hydroquinone (Upper panels). Mean tail moments were calculated in the same cells (Lower panels). Data represents Mean ± SE from at least 50 cells in each group. D: Immunofluorescence assay of γH2AX and 53BP1 in Hydroquinone-treated lymphoma cells. *<i>P</i><0.05 comparing with the FU cells.</p

<i>GSTT1</i> expression reduces tumorogeneity of PAH-treated lymphoma cells.

<p>A: Tumor formation in nude mice. Indicated amount of Hydroquinone treated cells were injected subcutaneously and tumorigenicity was reported as numbers of tumors formed per numbers of mice injected. B: Expression of GSTT1, Ki67 and MYC detected in tumor tissues by immunohistochemistry assay, as well as pCHK1, γH2AX and 53 BP1 by immunofluorescence assay. *<i>P</i><0.05 comparing with the FU cells.</p

Knock-down of <i>gstt1a</i> and <i>gstt1b</i> promotes lymphocyte proliferation exposed to BaP.

<p>A; WISH images showed the <i>rag1</i> expression in the thymus (arrows) of differently treated 5 dpf embryos. B: In situ analysis of <i>myca</i> at 5 dpf. The morphants showed increased expression of <i>myca</i> in microinjected <i>gstt1a</i> and <i>gstt1b</i> morpholino exposed to BaP (Left panels), semi-quantitative PCR showed similar expression pattern in embyos (Right panels). C: Ultrastructure of thymic lymphocytes from 5 dpf larvae exposed to BaP. Images represent results from three independent experiments and each group contains 30 morphants.</p

<i>GSTT1</i> deletion is related to decreased gene and protein expression and enhanced cell cycle progression.

<p>A: <i>GSTT1</i> gene expression assessed by quantitative real-time PCR in the <i>GSTT1</i>-deleting patients and normal controls. B: GSTT1 protein expression detected by tissue array. C: Geneset of cell cycle related proteins revealed by gene network and pathway analysis on microarray data of DLBCL and TCL.</p

<i>GSTT1</i> expression inhibits PAH-mediated lymphoma cell proliferation.

<p>A: Effect of <i>GSTT1</i> expression on cell proliferation. 3×10⁵ cells treated with normal saline or Hydroquinone were seeded and cell number was counted at 48 h by typan blue. Data represent Mean±S.E. of densitometric values from three individual experiments. B: EdU assay of Namalwa and Jurkat cells treated with normal saline or Hydroquinone. C: Cell apoptosis analyzed by flow cytometry. Histography indicates Mean±S.E. from three individual experiments. D: Key proteins of DNA damage and cell cycle detected by Western blot in Namalwa and Jurkat cells with or without Hydroquione treatment. *<i>P</i><0.05 comparing with the FU cells.</p

Induction of autophagy by valproic acid enhanced lymphoma cell chemosensitivity through HDAC-independent and IP3-mediated PRKAA activation

<p>Autophagy is closely related to tumor cell sensitivity to anticancer drugs. The HDAC (histone deacetylase) inhibitor valproic acid (VPA) interacted synergistically with chemotherapeutic agents to trigger lymphoma cell autophagy, which resulted from activation of AMPK (AMP-activated protein kinase) and inhibition of downstream MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling. In an HDAC-independent manner, VPA potentiated the effect of doxorubicin on lymphoma cell autophagy via reduction of cellular inositol 1,4,5 trisphosphate (IP3), blockade of calcium into mitochondria and modulation of PRKAA1/2-MTOR cascade. In murine xenograft models established with subcutaneous injection of lymphoma cells, dual treatment of VPA and doxorubicin initiated IP3-mediated calcium depletion and PRKAA1/2 activation, induced in situ autophagy and efficiently retarded tumor growth. Aberrant genes involving mitochondrial calcium transfer were frequently observed in primary tumors of lymphoma patients. Collectively, these findings suggested an HDAC-independent chemosensitizing activity of VPA and provided an insight into the clinical application of targeting autophagy in the treatment of lymphoma.</p