INTRODUCTION

Leukemia, a malignant hematopoietic tumor, is a cancer of the blood or bone marrow which is characterized by the abnormal proliferation of white blood cells and is the 6th most common form of human cancer worldwide (Tallman and Altman, 2009).

Acute Myeloid Leukemia (AML) is characterized by an accumulation of abnormal hematopoietic progenitor cells which exhibit the morphology of a certain stage of myeloid differentiation in the bone marrow and the peripheral blood (Grignani et al., 1998). The blocked differentiation restrains progenitor cells from maturing and subsequently undergoing apoptosis and it may also enhance their self-renewal capabilities. Unlike normal hematopoietic cells that require cell extrinsic signals to maintain metabolism and survival, leukemia cells often express constitutively active oncogenic kinases that promote these processes independent of extrinsic growth factors. When cells receive insufficient growth signals or glucose metabolism decreases and the self-digestive process of autophagy is triggered to degrade bulk cytoplasm and organelles. Given its potential function in metabolism and cell survival, manipulation of autophagy may provide a critical means to eliminate certain type of leukemia cells (Bernardi and Pandolfi, 2007).

The clinical picture of APL is characterized by a rapidly increasing bleeding tendency which is due to pronounced coagulation disorders and severe thrombocytopenia. The microgranular variant (AML M3v) is a morphologically distinguishable special APL form which is mainly associated with increased leukocyte counts. The APL constitutes a hematological emergency which requires immediate diagnostic confirmation and specific therapeutic measures (Nasr et al., 2008). Diagnosis requires the APL-specific chromosomal translocation t (15,17) (q 22, q 21) and/or the fusion gene PML/RARA (Nasr et al., 2009).

The introduction of All-Trans-Retinoid Acid (ATRA) in APL therapy doubled the cure rate relative to chemotherapy alone. Remission rates of 80-90% and rates of long-term survival exceeding 75%, were reached with the combination of ATRA and anthracycline-containing chemotherapy (Chomienne et al., 1996). Yet, more than 10% of the patients treated in studies die in the course of induction therapy, mostly as a result of bleeding complications. All-Trans-Retinoic Acid (ATRA), a derivative of retinoic acid, reverses the differentiation block of promyelocytic blasts and induces the development of mature neutrophils, associated with a regression of the coagulation disorders within a few days. The extraordinarily high efficacy of ATRA is APL-specific (Lallemand-Breitenbach et al., 2008).

Autophagy is a ubiquitous process in which “bad” cytosolic molecules damaged organelles or invaded pathogens are sequestered within double-membrane vesicles called autophagosomes that deliver their contents to lysosomes for degradation and/or recycling of the resulting macromolecules. Indeed, autophagosomes were found to specifically target damaged mitochondria. Peroxisomes and protein aggregates that lead to DNA damage and genomic instability, thereby maintaining intracellular organelle and protein homeostasis (Tatham et al., 2008).

Autophagy may also provide nutrients through lysosome degradation of intracellular components of mitochondrial oxidation. Autophagy is more frequently compromised in solid cancer cells compared with their normal counterparts and acts as a protective mechanism in response to both extracellular and intracellular stress (Shen et al., 2004). Heterozygosity for the autophagy-essential gene. Beclin1 led to greatly increased rates of tumorigenesis, possibly because of genomic instability when autophagy is reduced (Bence et al., 2001).

The PML/RARA oncoprotein is known to be prone to aggregation, a feature that makes it a good substrate for autophagic degradation. The aggregation-prone characteristics of PML/RARA are further substantiated by showing that this protein chimera is considerably more effectively extracted by buffers containing the protein denaturation reagent UREA compared with buffers containing the detergents SDS or Triton X-100 (Pankiv et al., 2007). Its ability to form protein aggregates is possibly attributable to the PML moiety of the fusion autophagy and proteasome-dependent degradation may thus cooperate in therapy-induced clearance of the APL-associated oncoprotein. In line with this finding, proteolytic crosstalk has been reported to clearly exist between the autophagy and ubiquitin-proteasome systems (Hu et al., 2009).

Furthermore, proteolytic degradation of PML/RARA by caspases and lysosomal proteases has also been reported, suggesting the existence of multiple proteolytic pathways with a potential to target PML/RARA for degradation. However, because autophagy appears to become markedly induced in the presence of all-trans retinoic acid and arsenic trioxide, this degradation pathway may have a determinant role in therapy-induced PML/RARA clearance. Thus, both all-trans retinoic acid and arsenic trioxide induce clinical remission in APL patients by stimulating mTOR-dependent autophagy and causing concomitant autophagic degradation of the APL-associated oncoprotein PML/RARA (Nedelsky et al., 2008). In patients with the characteristic morphological picture of an APL and severe coagulation disorders, the onset of therapy with ATRA is justified even before genetic analysis confirms the diagnosis. ATRA is administered continuously until complete remission is obtained, at maximum up to a period of 90 days. ATRA monotherapy achieves a complete remission in 80-90 (%) of the patients with newly diagnosed APL. However, long-term remissions are rare under ATRA monotherapy (Rowe and Tallman, 1997). The aim of this study was to investigate the effect of ATRA on the autophagic responses LC3-1, II and Beclin 1 proteins.

METERIALS AND METHODS

This study was conducted in Mansoura Pediatric Hospital and Oncology center in Mansoura University hospital. It included that 22 patients (13 females and 9 males) diagnosed as Acute Promyelocytic Leukemia (APL) (M3). The study also included 20 healthy subjects (12 females and 8 males) as a control selected to match the study group in age and gender. The Ethics Committee of Mansoura, Faculty of Medicine approved the study protocol. Informed consent was taken before testing. All patients were subjected to the following.

Patient’s history and physical examination and full laboratory investigation include the following:

Peripheral blood and BM findings: In the AML cases, clinical studies of the PB, BM cellularity, according to WHO classification 2008 (Swerdlow and Campo, 2008).

Immunophenotyping: We reviewed all available data, including Peripheral Blood (PB) smears, Bone Marrow (BM) aspirates, cytochemical staining immunophenotyping of cells was performed with flow cytometry against CD3, CD41a, CD14, CD34, CD33, CD20, CD5, CD10, CD19, CD64, CD11c, CD13, CD117, CD56, CD2, CD7, HLA-DR, cytoplasmic CD22, cytoplasmic myeloperoxidase, cytoplasmic CD3 and cytoplasmic CD79a (coulter, San Jose, CA) (Dunphy et al., 2004).

Conventional cytogenetics: Chromosomal analyses were performed by examining short-term cultures of BM specimens according to standard conventional cytogenetic protocols. At least 20 cells in metaphase were analyzed in each case. Clonal abnormalities were classified according to the 2009 International System for Human Cytogenetic Nomenclature guideline (Golomb et al., 1976).

Fluorescence in situ hybridization: Fluorescence in situ Hybridization (FISH) analyses were performed to confirm translocation and the presence of t (15,17). These FISH analyses used pellets of cells remaining after conventional cytogenetic studies. Slides for FISH were prepared by using cells harvested for conventional cytogenetics and processing them for FISH according to the manufacturer's guidelines (Abbott V lysis, Des Plaines, IL, USA). (Gurrieri et al., 2004). Analyses were performed on cells in either interphase or metaphasedual-fusion probe (Abbott V lysis) was used to identify t (15;17) and its variants (Rowley et al., 1977).

Western blotting for LC3-I and LC3-II formation expression: First extracted in ice-cold Triton X-100 containing lysis buffer (50 mM NaCl, 10 mM Tris, 5 mM EDTA, 1% TritonX-100+protease and phosphatase inhibitor cocktails), the whole cell extract was harvested in lysis buffer. Cell lysates were then centrifuged (14000 rpm) for 10 min and the supernatants (soluble proteins) collected. The remaining protein pellets were washed with Phosphate Buffered Saline (PBS) before extraction with an SDS-containing buffer (2% SDS, 1mM DTT, 50mM Tris+protease and phosphatase inhibitor cocktails) to obtain SDS. The protein concentration was analyzed using a BCA assay (Thermo Fisher Scientific, Fremont, CA, USA). Protein extracts were loaded and resolved on 12% gels (Pierce, Rockford, IL) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After the whole cell extract had undergone western blotting, the SDS-PAGE PVDF membrane was blocked with antibodies against Rabbit anti-LC3 (Cell Signaling, 1:5000), LC3 (Novus Biologicals, Littleton, CO, USA), PARP-1/2 (H-250) and then incubated with secondary antibodies, anti-rabbit IgG antibody (Jackson Immuno-research, West Grove, PA, USA). Actin or α-tubulin (Sigma-Aldrich) was used as an internal control (Klionsky et al., 2008).

Enzyme-link immunosorbent assay (ELISA) kit for Beclin-1 (BECN1): Measurement the level of Beclin-1 (BECN1) by ELISA using USCNK life science InsUSA.Cut off 1.25 (ng L^-1) (Sinha and Levine, 2008).

Statistical analysis: Results were collected, tabulated and statistically analyzed using statistical package version 16 (SPSS Inc., 2007). Continuous data was expressed as were eventually appropriate statistical tests. For normally distributed parameters group, comparison was made by use of paired and unpaired Student’s t-test. Mann-Whitney U-Test was used for non-normally distributed parameters. . In comparing 3 groups, one way ANOVA for statistical significance, were analyzed by the Mann-Whitney U-test (2 groups). Statistical significance was defined as p≤0.05 normally distributed data, presented as Mean±SD.