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Molecular Mechanisms of Intervertebral Disc Degeneration

Intervertebral disc degeneration is a well-known cause of disability, the result of which includes neck and back pain with associated mobility limitations. The purpose of this article is to provide an overview of the known molecular mechanisms through which intervertebral disc degeneration occurs as a result of complex interactions of exogenous and endogenous stressors. This review will focus on some of the identified molecular changes leading to the deterioration of the extracellular matrix of both the annulus fibrosus and nucleus pulposus. In addition, we will provide a summation of our current knowledge supporting the role of associated DNA and intracellular damage, cellular senescence's catabolic effects, oxidative stress, and the cell's inappropriate response to damage in contributing to intervertebral disc degeneration. Our current understanding of the molecular mechanisms through which intervertebral disc degeneration occurs provides us with abundant insight into how physical and chemical changes exacerbate the degenerative process of the entire spine. Furthermore, we will describe some of the related molecular targets and therapies that may contribute to intervertebral repair and regeneration.

Introduction
Over the past century, life expectancy has continued to increase, with an estimated 0.5 billion people worldwide aged 65 or older, and is projected to reach 1.5 billion people by 2050 1) . It is well documented that low back and neck pain increase with age, and are the 1 st and 4 th leading causes of disability, respectively [2][3][4] . Back pain is one of the most common orthopaedic conditions, and it accounts for 149 million missed worked days annually with an estimated loss of $90 billion in the United States, secondary to related disability 5) . Specifically, intervertebral disc degeneration (IDD) plays a substantial role in the generation of back pain, involving more than 50% of all cases [6][7][8] . In the elderly, decreased mobility is a confirmed predictor of loss of independence and increase in mortality. This makes the preservation and understanding the degeneration of healthy joints, especially intervertebral discs (IVDs), vital in our aging population [9][10][11] .
IDD refers specifically to the functional as well as the structural failure of the disc related to its cellular pathogenesis and extracellular matrix (ECM) modifications 12) . Causes of disc degeneration include aging, injury, genetics, and en-vironmental factors such as smoking, or a combination [13][14][15][16][17][18][19] . IDD is not exclusive to the older population, although the aged population and cases with degenerated disc appear to share similar changes [18][19][20][21][22] . The IVD is one of the first tissues to undergo degeneration in adults, with an average onset in the second decade of life and is known to be influenced by a combination of genetic, biological, aging, and physical chemical changes 20,21,23) . The exact pathophysiology of degeneration has not yet been completely delineated, but this review will focus on some of the known cellular changes and molecular pathways leading to IDD.

Characteristics of IVD
IVD are situated between two cartilaginous end plates of adjacent vertebra of the spine. They help in constructing a polyaxial cartilaginous joint that allows for flexibility, while also providing support 24,25) . The IVD is made of two major components: an annulus fibrosus (AF) and a nucleus pulposus (NP). The AF is composed of highly organized rings known as lamella, rich in collagen type I that surround the NP. These collagen fibers of the AF arranged in alternating angles of approximately 30-60 degrees serve to restrain the NP's circumferential stress during bending and twisting, while preventing lateral displacement and collapse of the NP 24,26) . The NP that is centrally located, is composed of approximately 80% water caused by the osmotic gradient of proteoglycans, primarily aggrecan, which consists of highly anionic glycosaminoglycan side chains of chondroitin and keratin sulfate. The NP also contains a loose and randomly arranged framework of sparse collagen type II and elastin fibers, which in combination with the proteoglycan, serves as a primary shock absorber defusing compressive stresses into the end plates and the annulus fibrosus 24,27) . In addition, the cartilage end plates, composed primarily of cells with a morphology similar to that of chondrocytes that produce a hyaline matrix, serve as an interface between the adjacent vertebral body and IVD 28) . As discs are predominantly avascular, the cartilaginous end plates are particularly important for nutrient and metabolite delivery to the disc by diffusion through its rich blood supply. This tenuous perfusion and low cellularity makes the disc susceptible to injury, in addition to the limited repair and accumulation of dysfunctional tissue 29,30) .
Degenerative changes lead to an altered spinal mechanical function with loss of elasticity and an increased stiffness 31) . Maintaining stability in the multiaxial motion of the spine is a complex interaction between all components of the spine working together counteracting compressive and tensile stress [32][33][34][35][36][37][38][39] . Degenerative changes of the spine that exacerbate the IDD process include facet cartilage erosion, weakening of supportive ligaments such as posterior and anterior spinal ligaments, and physiological muscle atrophy such as fatty infiltration [40][41][42][43] . Cells isolated from annular-puncture induced degenerative discs have shown unfavorable altered responses to mechanical loading and exaggerated response to inflammatory stimulus 44) . Studies in rats have indicated an increased IDD when they are imposed to an upright stance, which presumably alters the disc loading 45) . Other analyses have also made associations with long-term loading leading to loss of spinal mobility and decrease in disc height [46][47][48][49] . This loss of anatomical structure is the end result of damage-induced apoptosis, cellular senescence, and pathologic alterations to metabolism with the subsequent loss of standard cellular function and ECM structural support [50][51][52][53] .
It is known that the ECM of the NP and AF are vital to the biomechanical function and physiological state of the IVD 54,55) . The ECM of the NP contains only approximately 1% cells, based on its total volume. These cells produce structural matrix proteins, cytokines, growth factors, and proteases. All these products serve to maintain the balance between ECM production and degradation, while preserving biomechanical function 54,55) . Progressive proteoglycan loss with coinciding changes of low oxygen tension, free radical formation, changes in pH, and increased activity of aberrant proteolytic enzymes, leads to loss of disc height and compressive resistance. These changes result in a progressive disruption of the spinal motion leading to spinal instability and mechanical stress, compounding the degenerative process 56,57) .

Pathology of IDD
IDD has been defined by the loss of biologic structural support and function through the accumulation of degenerated molecules leading to inappropriate cellular reactions that exacerbate the pathogenesis 58) . These changes lead to a fibrous disc with dehydration of the NP, loss of disc height, and accumulation of granular debris. In addition, there is neovascularization in the peripheral AF and an increase in the number and size of fissures 20,59,60) . The cartilaginous end plates also undergo ossification and thinning with subsequent microfractures, bone sclerosis, and reduction in the blood supply. The reduction in perfusion followed by a decrease in nutrient supply and accumulation of cellular waste, all lead to an increase in the acidic environment that can negatively affect cell function 61-65) . The molecular mechanisms contributing to IDD that have been elaborated below result in cellular dysfunction through the accumulation of damaged proteins, altered intercellular communication, deregulated nutrient sensing, mitochondrial dysfunction, DNA damage, and interruption of tissue regenerative capacity with loss of progenitor cells (Fig. 1) 66-70) .

Degeneration of ECM in the NP
Typical ECM molecules in NP are proteoglycan aggregates, consisting of core protein, link protein, sulfated glycosaminoglycan (S-GAG), and hyaluronan, all encased within a collagenous fiber network. This ECM provides higher osmotic pressure to create swelling pressure capable of withstanding compressive loading stresses 71) . As discs degenerate and simultaneously dehydrate, the majority of the matrix evolves with decreased amount of GAG, aggrecan and elastin, and increased amounts of collagen and collagen crosslinking, fragmented aggrecan, and advanced glycation end productions (AGEs) 73) . This decreased amount of GAG chain lengths and link protein levels, while hyaluronan levels increase, are all thought to be a result of proteolytic and glycolytic damage 71-73) . Versican is another hyaluronanbinding protein that undergoes deterioration with the degeneration of discs. These non-aggregating proteoglycans appear to have a reduced functional ability compared to intact aggregates based on their size, charge density, spatial rigidity, and matrix interactions 71-76) . Syndecan-4 is a transmembrane heparan sulfate proteoglycan that also plays a significant role in IDD through intracellular signaling 77-79) . Specifically, dysregulated activities of syndecan-4 mediate matrix and aggrecan degradation by a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) function and matrix metalloproteinases (MMPs) expression e.g., MMP3 77-79) . The small leucine-rich repeat family of proteoglycans (SLRPs) typically play a significant role in structural support of the ECM. But with degeneration, SLRPs ap- pear to undergo proteolytic damage leading to a loss of their GAG content, negatively affecting the structural support of the ECM 71) . Specifically, biglycan and decorin, dermatan sulfate proteoglycans, interact with collagen type IV and type I/II respectively 71) . Others include disc collagen fibril associated SLRPs, feratin sulfate-containing fibromodulin and lumicans, which decrease and increase with age respectively, are thought to have an effect on disc matrix structure 71,80) . As mentioned above, discs contain a rich collagen network containing 80% of Type I and II, and 10-20% of type IV 71) . This collagen structure also undergoes proteolytic damage from dysregulated collagenase activity, resulting in weakened mechanical strength and the formation of non-enzymatic crosslinks between basic amino acids of collagen and reducing sugars 81,82) . As elaborated below, this proteolytic damage results in increased oxidative stress through AGEs, which increase throughout the disc with degeneration impairing collagen fibril formation 83,84) . To further support this concept, oxidative stress has been shown to decrease disc elasticity and alter the formation of secondary and tertiary structure of collagen molecules in mice models 31,83-85) . This increases their susceptibility to cleavage by MMPs, resulting in a deterioration of the biomechanical strength and structural integrity 31) .

Degeneration of ECM in the AF
Although the exact mechanism is not completely deline-ated, it is assumed that mechanical load, oxidative stress, genetics, inflammation, and DNA damage also contribute to a rupture of the AF through apoptosis and altered integrity with herniation of the NP 86,87) . Homeostasis in ECM, through regulation of catabolic and anabolic functions, have been shown to play an essential role in maintaining the integrity of the AF structure 88) . Specifically, with disc degeneration, this homeostasis is disrupted with excessive matrix catabolic activity 89,90) . Two apoptotic pathways in mammalian cells, the mitochondrial pathway and Fas/FasLigand pathway have both been shown to be involved in AF cell apoptosis regulation 91-93) . Islet amyloid polypeptide (IAPP), identical in composition to amyloid protein, participates in the regulation of glucose metabolism, reactive oxygen species, apoptosis, and inflammation 94-98) . It has been shown that the expression of IAPP, the calcitonin receptor, and receptor activity modifying protein decrease in AF cells with IDD 99) . This decrease in IAPP induces an increase in reactive oxygen species and intracellular calcium concentration along with a decrease in MMPs, all leading to cellular death 99) . Human samples, mouse models, and AF culture experiments have demonstrated that mechanical overload-induced IDD is mediated through the mitochondrial apoptotic pathway in AF cells 92) . In addition, abnormal loading on intervertebral disc resulted in a thickening and stiffening of collagen fibrils of the AF at the microscale and alteration of the collagen fibrils at the nanoscale. These changes likely lead to a change in the me-

DNA Damage
Proteins and other macromolecules can be degraded and replaced; however, this is not the case with DNA 66) . DNA requires repair mechanisms, and despite these rigorous mechanisms, cells still accumulate damaged DNA over time. It has been shown that inherited defects in genome maintenance mechanisms can lead to a variety of diseases with accelerated degeneration 67) . For example, DNA repair-deficient Ercc 1/D mice exhibit early onset of key IDD features, including loss of matrix proteoglycan, reduction in disc height, and increased cellular senescence 57,69) . DNA damage as a cause of IDD is further supported by exposure to genotoxic stress in humans and mice, including ionizing radiation and tobacco smoking that accelerate disc degeneration 101-104) .
Hyperosmolality has also been shown to induce DNA damage, through activation of the ATM/p53/p21 WAF1 pathway leading to the hypophosphorylation of the pRb protein and cell cycle arrest in the G1 phase of the cell cycle 105) . An increase in osmolality has been shown to lead to chromatin changes and DNA damage 106) . Although it is still unclear what level of hyperosmolality in NP is needed to induce DNA damage, cells of the NP are exposed to hyperosmolality levels up to 500 mOsm/kg H2O in vivo compared to < 300 mOsm/kg H2O in the majority of other tissues 105) .
Nutritional stress is another factor that can affect disc tissue leading to degeneration and cellular damage. Because discs are mostly avascular, the nutritional environment resides narrowly above the cellular requirements in the NP with low oxygen and glucose 107-109) . In addition, the disc maintains an acidic environment with high concentrations of lactate 110) . It has been shown that low O2 and low pH conditions can cause DNA damage and reduce proteoglycan and collagen synthesis 111) . This extreme environment puts the disc at risk to any additional stresses 112,113) .

Cellular Senescence
Cellular senescence, an important mechanism for the limiting of proliferation of potential cancer cells, has been described by cessation of cell proliferation due to the critical shortening of telomere length following successive replicative cell cycles 114,115) . Another type of cell senescence, stressinduced premature senescence (SIPS), results from accumulation of genomic and mitochondrial DNA damage. In addition, SIPS cells also acquire a senescence associated secretory phenotype leading to high amounts of secreted inflammatory cytokines and matrix proteinases causing a catabolic effect on neighboring cells and on the ECM, promoting degeneration ( Fig. 2) [116][117][118][119][120] . This pathologic disc matrix process is further supported by the observed amount of senescent cells which have been measured by an increased expression of senescent markers including senescence-associated bgalactosidase, p16 INK4A , and decreased telomere length. In addition, p16 INK4A has been shown to have a positive correlation with the expression of matrix metalloproteases e.g., MMP-13 and ADAMTs-5 [121][122][123][124][125] . Further supporting DNA impairment can lead to disc cellular senescence is the observation of elevated cellular senescence in the disc of DNA repairdeficient Erccl _/D genotoxin exposed mice 14,101,102) . Also, further supporting SIPS as a potential cause of disc degeneration are in vitro cell culture studies using H2O2 to simulate oxidative stress leading to DNA damage with a transformed, catabolic phenotype [126][127][128] . This leads to increased matrix degradation with high levels of MMPs and pro-inflammatory cytokines, with IL-1 as a predominant cytokine in the pathogenesis of IDD [126][127][128] .

Oxidative Stress and Deregulated Signaling
Oxidative inflammation is thought to be one of the main causes of molecular damage through exogenous and endogenous stressors (Fig. 3) 103,104) . The advanced glycation end productions (AGEs) including pentosidine and carboxymethllysine, further support oxidative damage as a source of molecular damage 80,129) . In the example of pentosidine, which cross links collagen molecules, there is evidence to suggest that it may play a role in increased collagen stiffness and fragility affecting the biomechanics 80,82, 129,130) . Also, oxidative post-translational modifications, i.e. protein carboxylation, have also shown an increase in protein fragmentation and aggregation leading to increased disc stiffness in a mice model 21) . Although disc cells reside in relative low oxygen tension environments, reactive oxygen species (ROS) are still generated through oxidative phosphorylation 131) . As disc degeneration and AF develop fissures with associated neovascularization, this leads to an increase in oxygen tension on otherwise hypoxic cells, further supporting the oxidative stress on the disc environment 131) .
ROS is detrimental to the structural and functional homeostasis of the disc by causing the damage of lipids, DNA, and proteins 132) . Hydrogen peroxide, identified in human NP tissue, along with peroxisomes detected in AF cells in vitro, further support discs cells as ROS generators 133,134) . With mitochondrion dysfunction as a known source of excessive ROS production, the role of mitochondrion-dependent ROS has been described in various disc cells including human and rat NP and AF cells [135][136][137][138][139] . And more specifically, exces-sive ROS production has been reported in degenerative discs of rats 140) . Several studies have shown hydrogen peroxide to down regulate the expression of collagen type II and aggrecan in both human and rat disc cells. 126,[141][142][143][144][145] In addition, proinflammatory cytokines leading to ROS overproduction have been shown to suppress matrix synthesis and increase the expression of matrix degradation proteases in human and rat disc cells 126,137,141,142,145,146) . ROS is also known to form positive feedback loops that enhance ROS production in disc cells 126,136,141,[145][146][147][148][149][150] .
Inappropriate and deregulated signaling (e.g. NF-κB and MAPK pathway) in addition to abnormal variations in cell fate (e.g., cellular senescence), dysregulated nutrient sensing, and mitochondrial dysfunction has also been reported in deteriorating tissues including IDD 70) . Elevated levels of the pro-inflammatory cytokine TNF-α, matrix proteoglycan degradative products, MMPs-including MMP-3, ADAMTS-5, have also been reported in degenerative disc suggesting an imbalance of matrix homeostasis [151][152][153][154] . Other phenotypic and functional changes that likely result from irregular responses to damage include depletion of disc matric proteoglycan, tissue dehydration, and altered disc load distribution. These distorted conditions lead to elevated necrosis, apoptosis, and senescence 112, 121,157,158) .
Potential sources of oxidative stress and DNA damage include inflammation and high glucose induced stress, as in diabetes 159) . NF-kB signaling is known to play a critical role in a cell's response to inflammation and damage, and an increase in its activity has been connected to IDD with accumulated oxidative stress 129,139,159,160) . Pharmacologic and genetic systemic inhibition of NF-kB activity has been shown to reduce associated IDD in a mouse model 141) . Symptomatic discs have been shown to have higher levels of proinflammatory cytokines, TNF-α, IL1β, IL-6, and IL-8, which are considered be associated with the NF-kB pathway 127,128,156,162,163 ) . Mitogen-Activated Protein Kinases (MAPKs), a family of signal transduction pathways, allow cells to respond to extracellular inputs, including inflammatory cytokines and environmental stress 164,165) . Specifically, the expression of p38 MAPK, a subfamily of MAPK, has been described in senescent AF cells 166) . Many components of the catabolic process (ex. MMPs, ADAMTSs, COX-2, PEGE2, iNOS, etc.) are dually regulated by MAPK and NF-kB, showing the overlap between the two signaling pathways 155) . As mentioned above, pro-inflammatory cytokines (IL-1β and TNF-α) activate MAPK pathways leading to catabolic molecules such as ADAMTs-4, MMP-3, and syndecan-4 146,[167][168][169] . This relationship may represent a tool to ease disc degeneration by blocking MAPK activation either with synesthetic or natural compounds such as glucosamine (Fig. 4) 146,170) . Another characteristic feature of disc degeneration is the formation of cell proliferation clusters in damaged areas, which is thought to be partly due to the overexpression of growth factor and receptors 171,172) . Indicating another role of MAPK in disc denegation is the fact that growth factors such as PDGF, IGF-1, and bFGF stimulate cell proliferation through extracellular signal-related kinases, another subfamily of MAPKs 173,174) .

Conclusion
The distinctive disc function that maintains stability and motion of a mechanically loaded structure in nutrient-poor, acidic, and hypoxic environment, offers an extraordinary opportunity to discover novel disc degeneration mechanisms.
As noted above, disc degeneration is a systemic process that does not occur in isolation and is influenced by the degeneration of adjacent spinal structures and systemic factors. Therefore, degeneration research of the whole spine is an imperative for the future direction of spine degeneration and regeneration research. And more specifically, therapeutic interventions will need to target the early phases of disc and spine degeneration prior to functional failure. Several recent studies have strengthened our understanding of intervertebral repair and regeneration. The in vivo performance of an acellular disc-like angle ply structure that mimics the native intervertebral disc was studied in a rat tail model, and it was noteworthy that native cells were able to infiltrate the angle ply structure while promoting tissue formation and restoring some biomechanical behaviors 175) . Mesenchymal stem cell injections, another way to induce regeneration, have been shown to increase the amounts of GAG accumulation when injected early into injured rat intervertebral discs 176) . Inorganic polysphates have been shown to promote proteoglycan accumulation in NP cells, even under hypoxic conditions 177) . In addition, bone morphogenetic protein 2 (BMP-2) and BMP-7 have been shown to stimulate the NP to produce aggrecan and collagen II in vitro and in organ culture models 178) . And as mentioned above, NF-kB inhibition results in the largest reduction of IL-1β, a pro-inflammatory catabolic cytokine 179) . With disc degeneration being a significant risk factor for associated pain and disability, the prevalence will continue to rise with our growing and aging population. This should help stimulate further research to develop safe and effective treatments for intervertebral degeneration and pathology.

Conflicts of Interest:
The authors declare that there are no relevant conflicts of interest.


s2fieldsofstudy: Biology Medicine

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Breast cancer resistance protein (BCRP) gene expression in a cohort of adult Egyptian patients with acute myeloid leukemia

Background Acute myeloid leukemia (AML), an aggressive clonal disease, is genetically heterozygous. The prognostic role of expression of Breast Cancer Resistance Protein (BCRP) gene, which behaves as a multidrug transporter, in adult AML is ambiguous. Objective The objective is to assess the level of mRNA expression of BCRP gene in newly diagnosed cytogenetically normal adult Egyptian AML patients; and to clarify its potential influence and association between therapeutic responsiveness and disease free survival. Methods The BCRP gene expression was evaluated by quantifying its mRNA using real time RT-PCR in fifty newly diagnosed cytogenetically normal adult AML patients and 20 healthy normal controls. The expression was evaluated in relation to clinical and prognostic factors, response to treatment and the survival rate. Results BCRP mRNA was over expressed in adult AML patients compared to controls. This study showed a positive statistical correlation between BCRP gene expression and the percent of CD34 expression. Statistical analysis did not reveal any association between BCRP expression level and chemotherapeutic responsiveness or disease free survival rate. Conclusion The significance of BCRP gene expression and its function in AML is very complicated, therefore more standardized clinical studies are needed.

Introduction
Acute myeloid leukemia (AML), clonal malignant aggressive hematological disease, caused by accumulating acquired genetic abnormalities in hematopoietic stem cells 1 . It includes diverse categories according to molecular and other cytogenetic abnormalities 2 . Several prognostic markers were described in AML, including genetic mutations, polymorphisms or over expression of specific genes 3 . One of these prognostic factors is the multidrug resistance (MDR) phenotype confirmed by the presence of transmembrane transporter proteins in leukemic cells 4 . The Breast Cancer Resistance Protein (BCRP), synonymously known as ATP-binding-cassette protein, is coded by a gene located on chromosome 4q22, it encodes a protein composed of 655-amino acids, when over expressed it creates a drug resistance phenotype in tumor cell lines 5 . Such abnormal phenotype is due to the pump out of a large spectrum of chemicals, including chemotherapeutic drugs, outside the cells by the transport function of this protein. So it's expression has a particular influence on cancer treatment 6 .
The overexpression of BCRP gene enables cancer cell lines to self-renew and differentiate, which are their main characteristic feature 5 . These cells play a crucial role in MDR, where their BCRP overexpression enable them to resist chemotherapy and survive after being exposed to chemotherapeutics. It is possible that by inhibiting BCRP, the cancer cells could be targeted and eradicated 5,7 . A limited number of clinical prognostic studies discussed BCRP mRNA expression in AML, with different selection criteria and interpretation methodology [8][9][10][11][12][13] . The aim of the current study is to assess the level of mRNA expression of BCRP gene by real time RT-PCR in 50 newly diagnosed cytogenetically normal adult Egyptian AML patients, trying to clarify its potential influence and its association between the therapeutic responsiveness and disease free survival.

Subjects and methods Study population
The present study was applied on 50 newly diagnosed cytogenetically normal adult AML patients. They were 26(52%) males and 24(48 %). females The age of the included patients ranged from 18 to 71 years with a mean of 41.2±15.9 years. Patients were selected over six months from National Cancer Institute, Cairo, Egypt. The Ethical Committee of National Cancer Institute approved the current research protocol. All procedures performed in the current study were in accordance with the World Medical Association Declaration of Helsinki and its later amendments. Twenty age and sex-matched healthy unrelated blood donors participated in this study as a control group.
After collection of written informed consent from all participants, patients were subjected to full clinical assessment in addition to bone marrow examination. Immunohistochemistry staining and immuno-phenotyping was carried out to confirm the diagnosis and for classification of AML. Chromosomal karyotyping, molecular analysis with special emphasis on; t (8;21), inversion of chromosome 16, Nucleophosmin (NPM) gene mutations and FMS-like tyrosine kinase 3 -internal tandem duplication (FLT3-ITD) were done. Patients diagnosed as promyelocytic leukemia (M3), were not included in the study due to different therapeutic modalities and prognostic criteria. AML patients were followed up over the period of induction chemotherapy and for twelve months.

BCRP m-RNA expression:
From cases and control samples, cellular RNA was extracted, then the complementary DNA was synthesized using QIAamp RNA blood Mini Kit, supplied by Qiagen, and High capacity cDNA Archive kit, provided by Applied Bio systems, respectively, the manufacture instructions were followed for each kit. Every PCR amplification reaction had a total volume of 25 μL which included: 1 μL of the cDNA, 12.5 μLTaqMan universal PCR master mix (Applied Biosytems, Inc.), 1 μL of each forward and reverse primers of BCRP with the following sequences;5'-CAGTACTTCAGCATTCCACGAT-3', and: 5'-GGCAGAAGTTTTGTCCCAAA-3'. In addition to 0.5μL of the probe of Taqman with the sequence of 5'-FAM-CATTATGCTGCAAAGCCGTAAATC-CA-TAMRA-3'. The Glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene was selected to be the housekeeping gene. The PCR reaction was programmed for; 10 min heating at 95°C, followed by 40 cycles of; 15 s heating at 95°C for denaturation and 1min at 60 °C for annealing and elongation. Each PCR run included negative control tube without addition of any cDNA template. The "comparative threshold method" (2 -ΔΔCT) was used to calculate the relative expression level of BCRP gene 13 .

Treatment protocol and response to therapy
The AML patients were treated in accordance to the protocol of the Department of Medical Oncology, Kasr Al-Ainy Faculty of Medicine, CairoUniversity. Induction and consolidation were the two implemented phases of therapy. The remission was induced by 7-3 protocol, where fit patients (who were <60-65 years old, and selected fit patients up to age 75 years) received intensive therapy. Whereas less fit patients (who were70-75 years and older, or younger patients with significant co-morbidities) received lower intensive therapy. To achieve this inductionphase, a combination of cytarabine and anthracycline or anthracenedione was recommended (cytarabine 100-200 mg/m 2 continuous IV infusion for 7 days, and for 3 days idarubicin 12 mg/m 2 /day, or daunorubicin 60-90 mg/ m 2 /day were added). All patients were assessed for risk of relapse. Specific drug protocol, for consolidation therapy, were recommended based on the risk of relapse of the patient, high-dose cytarabine 3 g/m 2 IV over 3 h every 12 h on days 1, 3 and 5 for 4 cycle.
After induction therapy, Complete remission(CR) was obtained when the patient had a cellular marrow with blast cells ≤5%, peripheral blood picture with a neutrophil count ≥1.5×10 3 /ml, platelet count≥100×10 3 /ml, and with no verification of leukemia in other sites. The patient was stated to be primary resistant if having cellular marrow with >5% blasts or verification of leukemia in other sites 14 .

Data analysis
Data arrangement and analysis were done using Statistical Analysis Systems, SAS vs8.02. The graphs were done using Harvard Graphics, vs4. The data were summarized according to their type, where mean and standard deviations (SD) or median and range were used for numeric data, while percentages were used for categorical data. Mann-Witney test was performed to compare numeric variables. The Chi-square test was applied to compare groups regarding the categorical data, while for small sample size Fisher's exact test was used.
The strength of association between two numeric variables was calculated using Spearman's correlation coefficient, where values close to 1 or -1 mean a perfect positive or negative correlation, respectively. The disease free survival (DSF) was estimated starting from the time of remission to relapse or death or loss to follow and was evaluated by the Kaplan and Meier method, while the log rank test was used for comparison. Each P-value < 0.05 was considered to be significant.

Results
The main laboratory data of AML patients were summarized in table 1. AML patients were categorized into two groups according to the relative expression of BCRP gene: 34 AML patients (68%) were considered as a high expressors, with relative expression level higher than that of the controls. Sixteen AML patients (32%) were considered as a low expressors, with relative expression level within the range of the normal controls. There was no statistical significant difference between the two patients'category regarding their clinical and laboratory data. After induction chemotherapy, Complete remission was achieved by 24 / 50 (48%) of AML patients. Among these cases, 15/24(62.5%) were high expressors and 9/24(37.5%) were low expressors. Twenty-six patients (52%) failed to achieve CR (12 patients showed resistance to induction treatment and 14 patients died during the course of induction). No statistical significant difference could be detected between high and low expressors groups according to the response of induction chemotherapy (Table 2). Follow up of AML patients for twelve months revealed that 18/24 (75%) patients who achieved CR after induction chemotherapy had a disease free survival(DFS)during the follow up period. Ten of them were high BCRP expressors, while eight were low BCRP expressors. Six pa-tients failed to achieve 6 months DFS, five of them were high BCRP expressors and one patient was low BCRP m-RNA expressor. There was no statistically significant difference noticed between high and low BCRP expressors regarding their DFS rate ( Figure 1).

Discussion
Several factors affect the prognosis of adult AML, such as the age of the patient, the strength of post remission therapy, beside the biologic characteristics of the disease. Karyotyping at the time of diagnosis, the existence of transmembrane transporter proteins which grant mul-tidrug resistance. The over-expression or the mutations affecting particular genes are also included in the factors affecting the disease outcome 4 . The present work revealed that BCRP mRNA transcript was not overexpressed in the peripheral blood of normal healthy donors. Abbott et al. , 8 found low levels of BCRP mRNA expression in normal samples, whether peripheral blood or bone marrow, which reflected the restricted BCRP expression to the rare repopulating hematopoietic stem cells 15 .
We chose the cutoff of expression level to be the highest reading of BCRP mRNA expression among the controls. Our results were in harmony with Abbott et al., 8 8 study, but this was lower than MDR cell lines. This finding can be explained by either the homogenous expression of BCRP by all leukemic cells, or that BCRP expression could be heterogeneous and restricted to a subpopulation of leukemic cells. Flow cytometry study of BCRP protein expression levels in adult AML found that the later scenario was more likely 8,9 .
In the current study, there was no statistically significant difference between AML patients with high BCRP expressors and low expressors regarding their clinical and laboratory data. Similarly,previous clinical studies on AML patients could not find an association between BCRP m-RNA expression and AML patient characteristics such as age , hemoglobin and total leucocytic count, nor among different FAB subtypes 7,9,10,13 . During the study of the possible association between BCRP expression and other prognostic markers, a significant correlation between the BCRP mRNA expression level and the percentage of CD34 expression on leukemic blasts was found. Otherwise there was no statistically significant correlation between BCRP gene expression and other clinical nor laboratory data of AML patients. This was in agreement with the results reported in van den Heuvel-Eibrink et al., study 12 who found a significant positive association between CD34 expression and BCRP gene expression. Abbott et al., 8 and Suvannasankha et al, 9 could not find an association between and BCRP gene expression and other clinical nor other laboratory data, even the CD34 and or /CD33 expressing AML sub-population. In Nasilowska-Adamska et al .,study 13 the expression of BCRP mRNA in blast cells, which were positive for CD34, was of a marginal significance .
Controversy about the expression of BCRP mRNA in human pluripotent stem cells has been encountered, where Zeng et al., 16 reported that these cells were less resistant to certain cytotoxic chemicals due to BCRP expression. However, with more sensitive techniques Sarkadi et al., 17 suggested that BCRP expression was at a relatively high level in the human undifferentiated stem cells, which highlights its role in protecting this valuable stem cells from the damage caused by toxins, drugs or hypoxia.
The present work did not conceal a significant difference in BCRP mRNA expression as regard the early treatment response nor to the respect of DFS of the patients. Our results were consistent with Abbott et al., 8 and Suvannasankhaet al., 9 . Furthermore, Uggla et al., 11 could not find a difference between treatment AML responders and non-responders patients as regards BCRP expression, but in the group of responding patients, those with uppermost BCRP mRNA expression showed significantly shorter overall survival.
On the contrary, the study of Nasilowska-Adamska et al., 13 found that, in a univariate analysis, the BCRP expression was associated with higher early relapse rate and significantly influenced the disease free survival in AML. Also van den Heuvel-Eibrink et al., 12 reported that BCRP expression was frequently up regulated in patients with relapsed AML. In addition, high BCRP expression level in AML patients has been associated with a relapsed or refractory state, lower complete response and shorter survival rates 10,18 . Damiani et al., 7,19 reported the same finding and stated that there was an up regulation in BCRP protein at relapsed AML samples when compared to newly diagnosed ones and it can identify AML patients with poor outcome even after stem cell transplantation.
As the biology of BCRP gene expression and its role in AML is much more complicated than in cell line models 8 , our study could not find a clear association between BCRP overexpression in adult AML cases and its possible contribution as a molecular predictor to the response of therapy. This could be attributed to the discordance between BCRP mRNA overexpression and subsequent translation into a functioning trans-membrane protein in a malignant and genetically complex disease such as AML 20 . Furthermore underlying genetic polymorphisms of the BCRP gene, as a part of the genetic heterogeneity in AML, could affect the expression levels with subsequent influence on the disease progression 6 multiple genetic, together with heterogeneous functional proteins, interactions need to be clarified in AML.
In order to better understand the clinical role of BCRP in AML, we recommend the estimation of the BCRP expression in a bigger cohort of AML patients, study genetic polymorphisms that could affect the expression levels, standardize several factors including patient selection, used chemotherapeutic drugs and development of consensus recommendations for BCRP detection. Moreover, longer follow up period will clarify the possible influence of BCRP expression on DFS and overall survival of the disease.


I. P. LEZHENINA, Yu. V. VASYLIEVA Megabruchidius dorsalis (Fåhreus, 1839) (Coleoptera: Chrysomelidae: Bruchinae) is a new adventive species in the Kharkiv Region (Ukraine)

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Lezhenina, I. P., Vasylieva, Yu. V. Department of Zoology and Entomology, Dokuchaiev Kharkiv National Agrarian University, P.O. ‘Dokuchaievske’, Kharkiv District., Kharkiv Region, 62483, UKRAINE; e-mail: iryna57lezhenina@gmail.com, vasilevaula952@gmail.com ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Received 03.12.2018 Accepted 17.12.2018 by Dr. V. L. Meshkova Published 21.12.2018 UDC 595.768.13:591.4/.5:591.9(477.54) DOI: 10.36016/KhESG-2018-26-2-3

I n t r o d u c t i o n .
The problem of alien species penetration into the territory of Europe attracts more and more attention, and the number of publications to adventive species increases significantly every year (Орлова-Беньковская, 2017) are devoted.
For the first time for Ukraine, an East Asian seed beetles indicated by V. V. Martynov and T. V. Nikulina in 2014 from the Donetsk Region (Avdeyevka) (Martynov, Nikulina, 2014). Later, in 2015, it was registered in Kyiv (Fursov, Nazarenko, 2015). Already in 2016, the Asian grain was found in all districts of the Donetsk Region (Мартынов, Никулина, 2016).
Information about this species for the Kharkiv Region is missing. Also, almost nothing is known about the biology of M. dorsalis in a new region. It is known that in Europe its larvae feed by the seeds of Gleditsia and Gymnocladus (György, 2007).
The a im of our work is study of M. dorsalis in the Kharkiv Region. M aterials a nd me tho ds. Material was collected in October-November 2018 in the stands of honey locust (Gleditsia triacanthos L.) in the arboretum of the Dokuchaiev Kharkiv National Agrarian University. Preliminary examination of the beans of the honey locust showed the presence of exit holes in seed coat, which prompted us to examine the beans. For analysis, 100 beans were collected on the ground and 100 beans from trees. The beans were collected randomly. They were also collected for phenological observations and placed into cotton bag, which is stored in an unheated room. Известия Харьковского энтомологического общества 2018, том XXVI, выпуск 2 I S S N1 7 2 6 -80 2 8 Вісті Харківського ентомологічного товариства 2018, том XXVI, випуск 2 The Kharkov Entomological Society Gazette 2018, volume XXVI, issue 2
Re sult s and disc ussion. The initial examination of survey of honey locust beans showed that they had round exit holes, as is characteristic for seed beetles.
Since the beans of honey locust are poisonous to most insects due to the presence of 5-hydroxypipecolinic acid and triterpenoid saponins (György, 2007), it was obvious that they were damaged by seed beetles of the genus Megabruchidius, which host is honey locust. Cutting of the beans revealed adults, larvae, and pupae of M. dorsalis have been found (Fig. 1-5).

F i g . 5 . Megabruchidius dorsalis, ♀ (photo by B. A. Kolomoets).
Megabruchidius dorsalis developed in 52 % of the beans harvested in the current year (collected from the trees in 2018). One or two specimens developed in one bean, no more than one larva in one seed (table).
As one can see from the Table, the beans, collected on the ground, were more infested -81%. In such beans, one to fifteen exit holes were found, and 19.1% of seeds were infested on average. It is obvious, that over three exit holes per bean is explained by the fact that the bruchid oviposits on the beans harvested in the previous years. In the article devoted to biology of the relative species M. tonkineus (Pic, 1904) (György, 2007), it is also mentioned, that bruchids often oviposit into the beans harvested in the previous years, lying on the ground. Counting the number of beans with over three exit holes we found out, that about 68 % beans were infested repeatedly. Известия Харьковского энтомологического общества 2018, том XXVI, выпуск 2 I S S N1 7 2 6 -80 2 8 Вісті Харківського ентомологічного товариства 2018, том XXVI, випуск 2 The Kharkov Entomological Society Gazette 2018, volume XXVI, issue 2


extfieldsofstudy: Biology Medicine

text:
Molecular Regulation of Differentiation in Early B-Lymphocyte Development

B-lymphocyte differentiation is one of the best understood developmental pathways in the hematopoietic system. Our understanding of the developmental trajectories linking the multipotent hematopoietic stem cell to the mature functional B-lymphocyte is extensive as a result of efforts to identify and prospectively isolate progenitors at defined maturation stages. The identification of defined progenitor compartments has been instrumental for the resolution of the molecular features that defines given developmental stages as well as for our understanding of the mechanisms that drive the progressive maturation process. Over the last years it has become increasingly clear that the regulatory networks that control normal B-cell differentiation are targeted by mutations in human B-lineage malignancies. This generates a most interesting link between development and disease that can be explored to improve diagnosis and treatment protocols in lymphoid malignancies. The aim of this review is to provide an overview of our current understanding of molecular regulation in normal and malignant B-cell development.

Introduction
The generation of B-lymphocytes in the bone marrow (BM) is a highly complex process guiding multipotent hematopoietic stem cells to become immunoglobulin-expressing B-cells. The differentiation process depends on the orchestrated activities of transcription factor (TF) networks and extracellular signals acting in conjunction to drive the expansion and maturation of progenitor populations. The process is complicated by the fact that cells must undergo Immunoglobulin (Ig) gene recombination and both positive and negative selection events to ensure proper functionality (reviewed in [1]). Even though much of our understanding of this developmental pathway is based on mouse models, there exist several similarities between mouse and human B-cell differentiation [2][3][4]. Furthermore, it is now evident that the same mechanisms that control normal B-lymphoid development in mice and humans are targeted in B-lymphoid malignancies (reviewed in [5]). The aim of this review is to provide an overview of our knowledge about developmental trajectories and regulatory networks in normal early B-lymphocyte development and their potential involvement in malignant transformation.

Resolving Developmental Trajectories in B-Cell Development
In order to understand the process controlling the generation of highly specified blood cells, it is of critical importance to identify and prospectively isolate cells at defined maturation stages. B-lymphocyte development has been suggested to proceed from the hematopoietic stem cell, through the lymphoid primed multipotent progenitor (LMPP) [6] stage, to generate a lymphoid-restricted common lymphoid progenitor (CLP) [7]. CLPs have the capacity to generate B-lineage-restricted B220 + Fraction A compartment [8], proceeding in differentiation to generate CD19 + cells. common lymphoid progenitor (CLP) [7]. CLPs have the capacity to generate B-lineage-restricted B220 + Fraction A compartment [8], proceeding in differentiation to generate CD19 + cells.
While the progenitor cells within the classical CLP compartment retain lymphoid linage potentials and display a reduced capacity to generate myeloid cells [7], the inclusion of additional surface markers in the staining protocols has revealed a molecular and functional heterogeneity within this population. Surface expression of Integrin α(2)β(7) (LPAM1) or CXCR6 identifies a subpopulation of cells with reduced B but preserved NK/T lineage potential [9], and BST2 expression identifies a dendritic cell population [10]. It is further possible to isolate a B220 + population with preserved combined B and T-lineage potential within the classical CLP compartment [11,12]. Hence, it has become increasingly clear that the CLP compartment is highly heterogeneous and likely harbors a variety of more or less lineage-restricted progenitors.
These findings could be seen as evidence that early B-cell development does not follow one distinct path but rather proceeds through multiple pathways whereby lineage potentials are lost in a more or less stochastic manner ( Figure 1). This model for lymphocyte development is supported by the finding that early thymic progenitors display combined T-macrophage potential but most have a limited ability to generate B-lineage cells [22]. Furthermore, the fetal liver contains cells with combined B-macrophage or T-macrophage potential [23]. Additional complexity in developmental trajectories in the fetal liver comes with the identification of B/T and B/NK bi-potent progenitors [9,24]. Hence, the difficulty of identifying CD19 − B-lineage committed progenitors could be a consequence of non-linear developmental paths not subject to the restrictions predicted from a hematopoietic tree (Figure 1).   While conventional surface marker expression did not allow for the prospective isolation of committed CD19 − B-cell progenitors, expression of a reporter gene under the control of the Igll1 (Lambda 5) promoter [25] allowed for the identification of B-lymphoid-restricted progenitors within the classical CLP compartment [26]. Despite the fact that the Igll1 gene, encoding one of the surrogate light chains [27], is not crucial for the earliest stages of B-cell development [28,29], the gene is transcribed in primitive progenitors serving as a marker for lineage commitment [30,31]. Continued analysis of the cellular heterogeneity within the CLP compartment identified the surface marker Ly6D as being expressed in a subpopulation of cells [32]. Transplantation experiments revealed that the Ly6D + "CLP" population was largely restricted to the production of B-lineage cells; subsequently, these progenitors were denoted B-lymphocyte progenitors BLPs [32]. While the BLP population displayed a minimal T-linage potential in vivo, in vitro differentiation analysis suggested that a fraction of the cells retained the ability to generate T-lineage cells in response to a strong Notch signal [12]. This indicates further heterogeneity within the BLP compartment. This heterogeneity is largely resolved using the expression of the surface markers GNDF family receptor α2 (GFRA2) and bone marrow stroma cell antigen 1 (BST1), to prospectively isolate BLP1, BLP2, and BLP3 cells displaying a progressive degree of commitment to B-cell development [33] (Figure 1). Even though the progenitor populations defined by GFRA2 and BST1 displayed variable levels of B220, their gene expression patterns as well as functional analysis suggest that they represent highly similar or even identical developmental stages. These findings would be in line with observations made using multiparameter molecular analysis of human B-lineage development suggesting a hierarchical model based on progressive and ordered loss of lineage potentials ( Figure 1) [34].
While CD19 expression marks stably committed B-lineage progenitors, this population is complex and can be further subdivided into defined developmental stages. The most immature cells express KIT, CD127 (IL7Ra), CD49E, CD11A, CD54, and CD43 [17,18,35,36], while CD25 (IL2Rα) is restricted to cells with a functional pre-BCR [17]. The expression of CD2 resembles that of CD25 because it is restricted to cells with cytoplasmic immunoglobulin heavy (IgH) chains [37]. Despite that surface antigens are most useful in the identification of progenitor stages in early B-cell development, a degree of heterogeneity can be observed even within a defined developmental stage [18]. This argues for the use of multiple markers for the identification of any given progenitor population. In all, the development of protocols for the prospective isolation of defined progenitor compartments provides a detailed map of the developmental trajectories in B-cell development generally compatible with the hematopoietic tree.

Transcription Factor Networks Regulating Early B-Cell Development
With our increasing understanding of the developmental trajectories in early B-cell development, it has been possible to explore molecular interplay at defined developmental stages. While lineage-specific gene expression is often associated with functional lineage commitment, it has been reported that the expression of lineage-associated genes can be detected already in multipotent progenitors [38,39]. These early transcriptional programs are suggested to be associated with defined cell fates [40], indicating the existence of functional lineage priming in non-committed progenitors ( Figure 2). Lymphoid lineage priming is mainly observed as activation of genes expressed in both B-and T-cells, including Rag1 and Dntt [39,41]. The activation of these genes is dependent on the TFs TCF3 [42][43][44], IKZF1 (IKAROS) [45], SPI1 (PU.1) [46,47], and MYB [48] acting in a concerted manner to promote the development of lymphoid progenitors. While SPI1 and IKZF1 create a regulatory loop controlling lymphoid versus myeloid cell fate [49], TCF3 initiates the B-lineage-restricted transcriptional program by activation of the FoxO1 gene [50] (Figure 2). FOXO1 acts in a feed-forward loop with the TF EBF1 [51] to activate the transcription of B-lineage genes during B-cell specification [52][53][54]. The ability of EBF1 to coordinate the activation of transcriptionally inactive genes in epigenetically silenced chromatin [55,56] is likely a consequence of EBF1 associating with chromatin remodeling complexes, thereby directly impacting the structure and the epigenetic landscape [57,58]. Despite the LY6D + "CLP" compartment being intact or even increased in the absence of EBF1 [59,60], the GFRA2 + compartment is dramatically decreased [33], the transcription of B-lineage-restricted genes is lost [51,52], and the cells are not properly lineage-restricted [60]. This highlights the essential role of EBF1 in B-lineage specification.
genes is lost [51,52], and the cells are not properly lineage-restricted [60]. This highlights the essential role of EBF1 in B-lineage specification. Even though EBF1 has the ability to repress genes associated with alternative cell fates [61,62] and the loss of EBF1 in B-lineage cells results in plasticity [63], stable lineage commitment depends on the TF PAX5. Despite a large portion of the B-lineage-restricted transcriptional program being activated in the absence of PAX5 [64,65], the progenitor cells are not stably committed to B-lineage cell fate [65][66][67][68][69]. In vitro differentiation or transplantation of PAX5-deficient pro-B cells, as well as deletion of the Pax5 gene in B-lineage cells, results in the formation of both myeloid and T-lineage cells in vivo and in vitro [65][66][67][68][69][70][71]. Lineage restriction is likely achieved through direct repression of target genes such as Colony-stimulating factor receptor 1 (Csf1r or c-fms) gene [72] and Notch1 [73]. Even though this suggests that lineage specification can be separated from commitment [65], the finding that PAX5 is a direct EBF1 target [74] links these two processes in normal development. PAX5 and EBF1 target control elements in the Ebf1 gene [75,76], creating a second regulatory loop and resulting in functional lineage commitment (Figure 2). The importance of the reciprocal regulation and collaboration between EBF1 and PAX5 is highlighted by the finding that normal as well as malignant pro-B cells carrying trans heterozygote mutations in the Ebf1 and Pax5 genes display lineage plasticity [77,78].
In addition to their function in stable lineage commitment, EBF1 and PAX5 are critical regulators of genes encoding proteins forming the pre-B cell receptor (pre-BCR) [54,[79][80][81][82]. This receptor is formed as a newly generated IGH chain complex with surrogate light chains IGLL1 (λ5) and VPREB as well as the signal transduction proteins CD79α and CD79β (reviewed in [1,83]). Combined signaling through the pre-BCR and the IL7 receptor stimulates a proliferative burst [84] and causes a reduction in RAG protein levels [85]. While the proliferative burst is of great importance for the expansion and overall production of B-lineage cells, progressive development and Ig light chain rearrangement depend on the cells exiting the cell cycle [86,87]. This maturation step is suggested to depend on a pre-BCR-mediated activation of a regulatory network involving interferon regulatory factor (IRF4) and PAX5 [88]. PAX5 targets and activates the Ikzf3 gene [89], encoding a TF suggested to collaborate with IKZF1 to repress genes encoding surrogate light chain components [90]. This results in a reduction of pre-BCR levels on the surface of the pro-B cell, reducing the proliferative signal. IRF4 has a somewhat different role in this process since it collaborates with the transcription factor FOXO1, stabilized by the pre-BCR signal [88], to drive differentiation and reactivate Rag gene expression critical for recombination of the Ig light chain genes [91][92][93]. Furthermore, IRF4 increases the expression of the CXCL12-responsive chemokine receptor CXCR4 [87]. This has been suggested Even though EBF1 has the ability to repress genes associated with alternative cell fates [61,62] and the loss of EBF1 in B-lineage cells results in plasticity [63], stable lineage commitment depends on the TF PAX5. Despite a large portion of the B-lineage-restricted transcriptional program being activated in the absence of PAX5 [64,65], the progenitor cells are not stably committed to B-lineage cell fate [65][66][67][68][69] [65][66][67][68][69][70][71]. Lineage restriction is likely achieved through direct repression of target genes such as Colony-stimulating factor receptor 1 (Csf1r or c-fms) gene [72] and Notch1 [73]. Even though this suggests that lineage specification can be separated from commitment [65], the finding that PAX5 is a direct EBF1 target [74] links these two processes in normal development. PAX5 and EBF1 target control elements in the Ebf1 gene [75,76], creating a second regulatory loop and resulting in functional lineage commitment ( Figure 2). The importance of the reciprocal regulation and collaboration between EBF1 and PAX5 is highlighted by the finding that normal as well as malignant pro-B cells carrying trans heterozygote mutations in the Ebf1 and Pax5 genes display lineage plasticity [77,78].

. In vitro differentiation or transplantation of PAX5-deficient pro-B cells, as well as deletion of the Pax5 gene in B-lineage cells, results in the formation of both myeloid and T-lineage cells in vivo and in vitro
In addition to their function in stable lineage commitment, EBF1 and PAX5 are critical regulators of genes encoding proteins forming the pre-B cell receptor (pre-BCR) [54,[79][80][81][82]. This receptor is formed as a newly generated IGH chain complex with surrogate light chains IGLL1 (λ5) and VPREB as well as the signal transduction proteins CD79α and CD79β (reviewed in [1,83]). Combined signaling through the pre-BCR and the IL7 receptor stimulates a proliferative burst [84] and causes a reduction in RAG protein levels [85]. While the proliferative burst is of great importance for the expansion and overall production of B-lineage cells, progressive development and Ig light chain rearrangement depend on the cells exiting the cell cycle [86,87]. This maturation step is suggested to depend on a pre-BCR-mediated activation of a regulatory network involving interferon regulatory factor (IRF4) and PAX5 [88]. PAX5 targets and activates the Ikzf3 gene [89], encoding a TF suggested to collaborate with IKZF1 to repress genes encoding surrogate light chain components [90]. This results in a reduction of pre-BCR levels on the surface of the pro-B cell, reducing the proliferative signal. IRF4 has a somewhat different role in this process since it collaborates with the transcription factor FOXO1, stabilized by the pre-BCR signal [88], to drive differentiation and reactivate Rag gene expression critical for recombination of the Ig light chain genes [91][92][93]. Furthermore, IRF4 increases the expression of the CXCL12-responsive chemokine receptor CXCR4 [87]. This has been suggested to stimulate migration of the pre-B cells to a micromolecular niche with low levels of Il7 to further reduce the proliferative signal [87]. Hence, B-cell development is driven by an intricate interplay between stage-specific regulatory TF networks that orchestrate the differentiation process.

Transcription Factor Networks Link Development to B-Lymphoid Malignancies
While these TF networks clearly play crucial roles in normal B-cell development, it is becoming increasingly clear that they are closely connected to malignant transformation (reviewed in [5]). This is because genetic alterations in the PAX5, EBF1, or IKZF1 genes are observed in a majority of B-ALL patients [94][95][96]. Even though there are reports of translocations of TF coding genes resulting in deregulated expression [97,98] or formation of fusion proteins [99][100][101][102][103], the most common genetic alterations result in partial inactivation of one or several TFs [94][95][96]. Even though inherited point mutations in PAX5 have been reported to result in increased leukemia incidence [104], the reduced functional TF activity more commonly depends on the inactivation of the TF genes via somatic heterozygote mutations and deletions [94][95][96]. The importance of TF dose for normal blood cell development in mice is well established because heterozygote inactivation of the Myb [48], Spi1 [105], Bcl11a [106], Ikzf1 [107], or Ebf1 [108] gene results in disturbances in B-cell differentiation. Despite the fact that the heterozygote inactivation of the Pax5 gene in mice does not appear to result in any dramatic developmental block [109,110], it has been suggested to result in alterations of cellular metabolism that may promote transformation [111]. Furthermore, PAX5 deletions are often found in combination with complex karyotypes and other genetic aberrations, including recurrent translocations like t(12;21)(p13;q22) (ETV6-RUNX1) or t(1;19)(q23;p13) (TCF3-PBX1) [94,96]. PAX5 mutations are also found in combination with genetic alterations in other TFs such as IKZF1 and EBF1 [94,96], likely augmenting the effect of a reduced PAX5 dose. Interestingly, the phenotypic changes in B-cell development in mice carrying heterozygote inactivation of a TF gene are often exacerbated upon combined targeting of two TFs. Combined heterozygote inactivation of Ebf1/Tcf3 [108], Ebf1/Runx1 [112], or Ebf1/Pax5 [113] results in more dramatic phenotypes than what is observed in the single mutants. This highlights the importance of functional and correctly balanced TF networks in B-cell development.
Even though leukemia is generally considered to be confined to one defined hematopoietic lineage, about 7% of patients display a more complex disease [114,115]. These malignancies are denoted as acute leukemia of ambiguous lineage (ALAL). ALAL can be manifested either as bi-lineal leukemia, involving several lineages, or bi-phenotypic disease, with expansion of cells displaying combined expression of normally lineage-restricted surface markers [114][115][116]. It has been reported that the level of PAX5 regulates the formation of bi-phenotypic leukemia [117] and that B-ALL cells carrying mutations in Pax5 can be converted into other lineages with preserved malignant features [78,118]. Furthermore, in MYC-induced lymphoma, oscillations in EBF1 and PAX5 levels result in lineage plasticity [119]. Additionally, dramatic phenotypic changes with preserved cytogenetic features have occasionally been reported from patients experiencing relapse of disease [120], further challenging the idea that leukemia is restricted to a given lineage of cells. While this has been considered uncommon in clinical practice [114,115], novel treatment protocols may reveal a higher degree of complexity in B-ALL.
Recently it was reported that 13 out of 20 patients relapsing after treatment of B-ALL with genetically manipulated T-lymphocytes (chimeric antigen receptor (CAR-T) cells) targeting CD19 developed CD19 negative leukemia even at the clonal level [121,122]. This makes lineage plasticity a central mechanism for resistance development upon targeted treatment of leukemia. Hence, disruptions in transcription factor networks in leukemia may impact not only the transformation process per se but may also underlie the development of resistance to lineage-targeted therapies.

Integration of External Signals and Transcription Factor Networks in Early B-Cell Development
Even though intrinsic cell events such as regulatory loops created by TF networks are of critical importance for B-cell differentiation, the normal development and expansion of progenitors depend on extracellular signals in the microenvironment. While some of these signals are shared with other hematopoietic progenitors, others are restricted to lymphoid progenitor compartments. Among the former are Kit ligand (Steel factor, Stem Cell Factor SCF), acting via the receptor tyrosine kinase cKIT [123]. This receptor is expressed in a variety of hematopoietic progenitor cells including the multipotent hematopoietic stem cells [124], myeloid progenitors [125,126] as well as the CLPs [7]. Subsequently, disruption of this signaling pathway results in defective formation of multiple hematopoietic lineages [127][128][129]. The expression of cKIT is rather restricted in B-lymphoid progenitors, but a substantial fraction of the pro-B cell compartment retains this surface receptor as well as an ability to respond to the cytokine [130]. CXCL12 is another broadly acting cytokine involved in the homing of cells to specific niches in the BM [131,132]. The protein acts via its surface receptor CXCR4 and both the ligand and the receptor are crucial for normal homing of hematopoietic progenitor cells [131,132], including B-cell progenitors [133]. The expression of CXCL12 is restricted to specific subpopulations of stroma cells in the BM [131], contributing to the ability of this chemokine to act as an organizer of the BM microenvironment.
B-cell development is also influenced by cytokines with more restricted activity. These include FLT3 ligand (FL) acting through the FLT3 receptor expressed in the earliest lineage-restricted progenitor cells. Both the LMPP [6] and the CLPs [134] express FLT3; however, upon progression of B-cell development, the expression is downregulated as a consequence of the Flt3 gene being repressed by PAX5 [135]. Ectopic expression of the FLT3 ligand causes alterations in blood cell development [136] and disruption of this signaling pathway results in reductions in LMPPs and CLPs [137]. However, this occurs without dramatic changes to the peripheral CD19 + B-cell compartments [137]. The phenotype is exacerbated when combined with inactivation of Il7 signaling since this results in a complete block in BM B-cell development [138]. In line with the idea that the Il7R is expressed on CLPs with all lymphoid lineage potentials [7], lineage tracing analysis suggests that all B-and T-lymphoid cells as well as a substantial portion of the NK cells in the adult mouse have a history of Il7 expression [139]. The IL7 receptor is expressed on B-cell progenitors and a deficiency in either the receptor [140] or the ligand [141] results in a dramatic impairment in B-cell development already in the B-cell-restricted CD19 − compartments in the mouse BM [60,142]. The Il7 receptor α (IL7Rα) chain is also a component of the receptor for thymic stromal lymphopoietin (TSLP), a cytokine acting via the Il7Rα and a specific TSLP-receptor [143]. IL7 and TSLP appear to be functionally redundant since ectopic expression of the latter largely rescues B-cell development in IL7-deficient mice [144].
Many cytokines would appear to be permissive, stimulating proliferation and reducing apoptosis in the B-cell progenitors rather than driving development to a specific lineage in an instructive manner. However, while T-cell development can be largely rescued by overexpression of BCL2 in mice deficient in IL7 signaling, this does not fully rescue B-cell development [145,146]. This suggests partially distinct functions for IL-7 signaling in the formation of different lymphoid lineages. One potential explanation for this could be that STAT5 activation, resulting from IL7R signaling [147], induces Ebf1 transcription, potentially driving the progenitor towards B-cell fate [148]. Furthermore, ectopic expression of EBF1 partially rescues B-cell development in mice lacking IL7 [148] or the BTB/POZ domain transcription factor ZBTB17 (MIZ1), crucial for functional IL7 signaling [149]. While this would suggest that IL7 has unique functions in the induction of the genetic program in early B-cell progenitors, the finding that ectopic expression of FL can rescue B-cell development independently of IL7 argues for a more permissive function [150]. Furthermore, the developmental block imposed by conditional deletion of STAT5, a key mediator of Il7 signaling [147], can be partially rescued by ectopic expression of anti-apoptotic proteins [151]. In all, it would appear that the IL7 signaling pathway has both permissive and instructive components, as supported by the finding that deletion of the pro-apoptotic protein BIM rescues survival but not differentiation of B-cell progenitors in Il7-deficient mice [152].
Despite the function of Il7 in human B-cell development being somewhat disputed [153], inactivating mutations in the common gamma chain results in severe combined immunodeficiency in humans [154] and activating mutations in the IL7 signaling pathway are commonly detected in human malignancies [155]. Interestingly, heterozygote deletion of Pax5 or Ebf1, in combination with transgenic expression of a constitutive active STAT5, causes a synergistic increase in the formation of B-lineage leukemia [156]. Hence, the interplay between TF networks and extracellular signals is critical for normal B-cell development and disturbances may result in impaired immune response or lymphoid malignancies.

Concluding Remarks
While the detailed understanding of maturation pathways is often considered a subject mainly relevant to developmental biology, our increased understanding of molecular events involved in malignant transformation highlights the relevance of cell differentiation in malignant transformation. Understanding developmental trajectories can be important for diagnosis since, even though leukemia is caused by expansion of progenitor B-cells, the heterogeneous expression of surface IG suggests that leukemia can reside in both the pro-and pre-B cell compartments [114,115]. The use of more advanced FACS staining protocols may resolve an even higher heterogeneity and possibly better classify leukemia in both the CD19-positive and CD19-negative (ALAL) groups. Furthermore, it is becoming increasingly clear that the regulatory networks that drive normal development are targeted in the transformation process. This is knowledge that can be explored to identify novel diagnostic and therapeutic approaches. Additionally, the understanding of the molecular regulation of lineage stability can be used to predict the risk of relapse through lineage conversion in association with targeted therapies. Hence, it can be predicted that basic developmental biology will become of increasing importance for the improvement of modern cancer care in the near future.
Funding: This work was supported by grants from the Swedish Cancer Society, the Swedish Childhood Cancer Foundation, the Swedish Research Council including the Stem Therapy and BioCare programs, Knut and Alice Wallenberg's Foundation, and a donation from Henry Hallberg.