Analysis of Oxford medial unicompartmental knee replacement using the minimally invasive technique in patients aged 60 and above: an independent prospective series

We present the outcome of an independent prospective series of phase-3 Oxford medial mobile-bearing unicompartmental knee replacement surgery. Eight surgeons performed the 154 procedures in a community-based hospital between 1998 and 2003 for patients aged 60 and above. Seventeen knees were revised; in 14 cases a total knee replacement was performed, in 3 cases a component of the unicompartmental knee prosthesis was revised, resulting in a survival rate of 89% during these 2–7 years follow-up interval. This study shows that mobile-bearing unicompartmental knee replacement using a minimally invasive technique is a demanding procedure. The study emphasises the importance of routine in surgical management and strict adherence to indications and operation technique used to reduce outcome failure.
Introduction
Modifications over the past 15 years have improved unicompartmental knee replacement surgery, as indicated in recent reports on the procedure [1–4]. The designers [5] (the originators) of the Oxford unicompartmental knee prosthesis (Biomet, Warsaw, IN) reported in 1998 a 97.7% cumulative survival rate of 10 years. An independent series with a 15-year survival analysis claimed a 94% cumulative survival rate [6]. The outcome was dependent on proper patient selection, surgical techniques and implant design, [4, 7] and the results have been attributed to improvements in these factors. The procedure is now performed through a short incision from the medial pole of the patella to the tibial tuberosity. Using this approach, there is little damage to the extensor mechanism, the patella is not dislocated, and the suprapatellar synovial pouch remains intact. As a result, patients recover more quickly. Patients achieve knee flexion, straight leg-raising, and independent stair-climbing three times faster than after total knee replacement (TKR) and twice faster than after open unicompartmental knee replacement surgery [8]. The minimally invasive procedure has been shown to be reliable and effective [9].
Because of the favourable published clinical results, surgeons at the Martini Hospital in Groningen, the Netherlands, began using the Oxford knee prosthesis in 1998. The goal of this independent prospective study for patients 60 years of age and above was to compare and evaluate the clinical midterm results of the Oxford phase-3 unicompartmental knee replacement using the minimally invasive technique in a community hospital.
Materials and methods
Between December 1998 and 2003, 154 successive Oxford unicompartmental knee replacements were performed in patients 60 years of age and above (Table 1). Of these, 132 patients underwent unilateral surgery, 10 patients underwent bilateral surgery on separate occasions, and 1 patient underwent concomitant bilateral surgery in the same OR session. There were 86 women; the average patient’s age was 69.2 years (range 60–93 years). All patients gave informed consent before their inclusion in this prospective study. Five patients had secondary osteoarthritis because of previous trauma. The remaining patients had primary osteoarthritis.
Table 1Oxford phase-3 unicompartmental knee replacementCriteriaResultsNumber of patients132Number of knees154Left/right knee (%)53.8/46.2Age (mean/range, in years)69.2 (60–93)Gender (M/W)57 (40%)/86 (60%)BMI30.7 ± 4.9Follow-up range2–7 years
Standardised anteroposterior radiographs were obtained with the patient in a weight-bearing position (standing), and lateral radiographs were obtained with the patient in a non-weight-bearing position (the patient lying horizontally). The radiographs were examined for loosening or radiolucency around the femoral and tibial components, and the anatomical axis of the limb was measured. The imaging criterion for no increased risk for loosening of the bone was a <2 mm thick radiolucent line [10]. The presence of osteoarthritic changes in the nonreplaced compartment was graded according to the Ahlback classification of osteoarthritis (Table 2) [11]. These procedures were performed by eight senior staff surgeons over the study period. Mean preoperative range of motion was 122.9 ± 8.9° of flexion and −0.7 ± 4.5° of extension.
Table 2The Alhback radiological scoring system for estimating the severity of OAGrade 0 Normal Grade 1 Joint narrowing Grade 2 Joint obliteration Grade 3 Bone destruction <5 mm Grade 4 Bone destruction >5 mmGrade 5 Subluxation
The results (preoperative, intraoperative, and follow-ups at 3 months, 6 months, and 1 year) were prospectively recorded with a historical record, procedure record, Knee Society score, SF-36 questionnaire (short form consisting of 36 questions), and the Western Ontario McMaster (WOMAC) score. Knee Society score ratings of excellent (90–100 points) and good (80–89 points) indicated success. The preoperative scores of the patients are presented in Table 3.
Table 3Scoring results of the non-revised patientsScoringResultsKnee society scoreKnee score Preoperative39.2 (SD 18.2) Postoperative89.4 (SD 14.0)Function Preoperative55.8 (SD 14.3) Postoperative77.1 (SD 24.7)Total score Preoperative47.6 (SD 12.3) Postoperative83.4 (SD 16.8)WOMAC scorePain Preoperative50.3 (SD 18.7) Postoperative78.6 (SD 21.5)Stiffness Preoperative51.2 (SD 22.6) Postoperative71.2 (SD 20.8)Function Preoperative50.6 (SD 20.7) Postoperative76.2 (SD 20.4)SF-36 questionnaireFunction Preoperative35.7 (SD 17.6) Postoperative56.1 (SD 24.5)Physical Preoperative28.2 (SD 37.2) Postoperative57.2 (SD 44.3)Pain Preoperative32.7 (SD 19.2) Postoperative59.8 (SD 26.5)Health Preoperative63.7 (SD 22.2) Postoperative61.4 (SD 21.7)Social function Preoperative52.6 (SD 17.1) Postoperative64.5 (SD 17.6)Emotional Preoperative64.5 (SD 44.6) Postoperative70.5 (SD 40.7)Mental health Preoperative73.7 (SD 17.9) Postoperative75.1 (SD 18.8)SD standard deviation
Preoperative weight-bearing radiographs showed that the knees had an average femorotibial alignment of 2.4° of valgus (range 8°–3° of varus). Thirty-seven knees had grade-1 Ahlback osteoarthritis [11] in the lateral compartment on the preoperative radiographs, and one had grade-2 Ahlback osteoarthritis. The preoperative skyline view of the patellofemoral joint showed no bone loss with eburnation and longitudinal grooving in all the cases.
All medial compartment arthroplasties were performed using the minimally invasive technique and under tourniquet control. The discharge criteria were control of immediate postoperative pain and the ability to flex the operated knee to a minimum of 90° with no lack of extension. All complications and revisions were reported, and a revision was defined as any surgical procedure resulting in removal or exchange of any of the prosthetic components.
Results
At the time of follow-up, two patients who had no known revisions were lost for the follow-up. The remaining 130 patients were available for follow-up. At the final follow-up, June 2006, revision TKR was performed in 14 knees and a prosthetic component was exchanged in three knees. An overview of the revisions is given in Table 4.
Table 4Revisions of Oxford phase-3 knee replacement surgeryIncidenceRevision of a component of UKA3 Revision of the mobile bearing1 Revision of the femoral component and the bearing1 Revision of the tibial component and the bearing1Conversion to a TKR14Reason for revision to a TKA Inappropriate indication1 Misalignment and loosening5 Infection1 Progression of osteoarthritis in lateral compartment4 Persisting anteromedial pain >1 year3
One bearing was replaced because of luxation after a hyperflexion trauma. A new bearing of the same size was inserted, and no recurrence of luxation was seen at follow-up. In another case of luxation of the bearing, the femoral component, and the bearing were changed 9 months after the primary surgery. The fixation of the femoral component in this case was insufficient. The multiple small drill holes were not made, and there was no cement in the large drill hole. With flexion, the loose femoral component moved distally, causing luxation of the bearing. The tibial component and bearing revision was performed seven months after the primary surgery because of misalignment of this tibial component. With flexion, there was impingement of the bearing with the tibial component, causing a clicking sensation and rotation of the bearing.
In one case, there was grade-2 Ahlback osteoarthritis [11] in the lateral compartment on the preoperative radiograph. This patient had no relief of preoperative pain, and the knee underwent TKR 18 months after the primary surgery. In five cases loosening of the components occurred; misalignment of the components is probably caused by impingement of the bearing. One patient had a deep Staphylococcus aureus infection, and a two-stage procedure was performed leading to a TKR. In four cases of revision, progression of osteoarthritis was seen in the lateral compartment with reported pain on the lateral side. These patients had a mean postoperative anatomical axis, femorotibial alignment of 18.6°. This overcorrection causes overloading of the lateral compartment with progression of arthritis in that compartment.
Three patients with persisting anteromedial pain underwent revision. In two cases, no cause was found, and in both pain persisted after TKR. In the third case, the synovial biopsy showed synovitis villonodularis pigmentosa, and after the TKR this patient was pain-free. Except for the two patients with persisting anteromedial pain, all patients with a conversion to TKA were pain-free. No special augmentations or revision prosthetic components were necessary in these procedures; there were no bone defects that required the use of particulate autograft or allograft, and primary cruciate-retaining TKA was used in the revisions.
Postoperative complications occurred after the primary unicompartmental knee replacements. One patient had a traumatic medial tibia plateau fracture 4 weeks postoperatively, which was treated conservatively. Another patient developed hemarthrosis that required extended hospitalisation; this was resolved with conservative treatment. There was one deep infection, and no deep venous thrombosis was reported.
At the time of the most recent follow-up, average flexion was 125.8 ± 13.8°, with two patients achieving <90° flexion. The average flexion deformity/extension was 0.3 ± 2.2°.
The postoperative scores of those patients who did not undergo revision (140 knees) at the latest follow-up are presented in Table 3. The Knee Society score total was 83.4. All three WOMAC scores improved. For the SF-36, the function, physical, and pain scores showed an improvement in the outcome; the other scores remained approximately the same.
The final follow-up radiographs showed an average anatomical axis, femorotibial alignment of 8.8° of valgus (range 4°–22° of valgus). The knees were corrected by an average of 6.4° (range 2°–14°). This relative overcorrection gives increased stress on the lateral compartment. Signs of osteoarthritis progression in the uninvolved tibiofemoral compartment on the radiograph at the last follow-up were noted in 43 knees (grade-1 Ahlback osteoarthritis in 39 knees and grade-2 Ahlback osteoarthritis in four knees). No grade-3 or -4 changes were noted. At final radiographic evaluation, no component showed evidence of loosening. No knees had >2 mm of tibial cement-bone radiolucency. There were no radiolucent lines seen at the posterior aspect of the femoral components.
Seventeen knees were revised, resulting in a survival rate of 89% in these 2–7 years of follow-up interval.
Discussion
The purpose of this prospective study was to evaluate midterm durability of Oxford unicompartmental knee replacement surgery for patients 60 years of age and older. We acknowledge that the present study has the limitations of a midterm follow-up. However, longer follow-up for this phase-III version with the minimally invasive technique is not possible, because the current version has been available only since 1998 [7]. Besides, most technical failures occur within the first 2 years [12].
In these 2–7 years of follow-up interval, 11% of unicompartmental knee arthroplasties in all patients needed revision—a survival rate of 89%. These results are considerably lower compared to the designer [5] series or the independent series [6].
The primary need for revision surgery could be attributed to indication and technical failures. Thirteen of the 17 revisions were probably related to human error, the remaining four are in one case a hyperflexion trauma and luxation of the bearing, one case with deep infection, and two cases with unexplained persisting anteromedial pain. Misalignment of the components was the primary cause of technical failure. With the minimally invasive technique, the visual field is restricted, making mobile-bearing unicompartmental knee replacement surgery a demanding procedure. Introduction of the minimally invasive option makes the terms surgical technique and pitfalls actual again.
For the remaining 113 patients (140 knees) who did not undergo revision, the Knee Society score, WOMAC and SF-36 questionnaires showed an improvement in the outcome. All three scores indicated less pain and improvement in function, as confirmed by an average clinical average flexion of 126° at the latest follow-up. The Knee Society score total of 83.4 indicates a successful outcome.
Over the 7-year period of our study, eight senior surgeons performed the operation with an average of <10 procedures a year per surgeon. All surgeons attended the instructional course organized by the designer group. There is no evidence for a learning curve in our study. The outcome should be attributed to the number of operations performed. As a result of the relatively low survival rate of this study, the number of senior surgeons performing the procedure in this hospital is now reduced to two.
Conclusion
Careful patient selection, surgeon experience, and proper instrumentation and surgical technique are important factors in mobile-bearing unicompartmental knee replacement surgery [13, 14]. For unicompartmental replacement surgery, long-term results are related to the number performed by the unit [14]. The surgeon should be well versed in the routine, indications, and technique of this procedure to minimise failure rates.

unicompartmental
knee
replacement
mobile bearing


New insights into purinergic receptor signaling in neuronal differentiation, neuroprotection, and brain disorders

Ionotropic P2X and metabotropic P2Y purinergic receptors are expressed in the central nervous system and participate in the synaptic process particularly associated with acetylcholine, GABA, and glutamate neurotransmission. As a result of activation, the P2 receptors promote the elevation of free intracellular calcium concentration as the main signaling pathway. Purinergic signaling is present in early stages of embryogenesis and is involved in processes of cell proliferation, migration, and differentiation. The use of new techniques such as knockout animals, in vitro models of neuronal differentiation, antisense oligonucleotides to induce downregulation of purinergic receptor gene expression, and the development of selective inhibitors for purinergic receptor subtypes contribute to the comprehension of the role of purinergic signaling during neurogenesis. In this review, we shall discuss the participation of purinergic receptors in developmental processes and in brain physiology, including neuron-glia interactions and pathophysiology.
Introduction
During the last two decades, evidence for the participation of ATP as neurotransmitter in neuronal signaling was collected by Drs. Surprenant [1] and Silinsky [2]. Purine-sensitive receptors were first classified as P1 G-coupled receptors which are activated by adenosine and P2 receptors, responding to stimulation of ATP [3]. Based on receptor cloning and studying of receptor-induced signal transduction, P2 receptors were divided into P2X receptors as ATP-gated ion channels and P2Y G protein-coupled receptors [4].
The expression of purinergic receptors has been identified during development and differentiation processes [5–10]. Nucleotides exert a synergic effect on cell proliferation in association with growth factors, chemokines, or cytokines in early stages of development [11–13] by parallel activation of the MAP kinase pathway and/or by transactivation of growth factor receptors [14, 15].
The complete role of ATP action in developmental processes still needs to be elucidated. It is known that ATP activates purinergic receptors resulting in many cases in increases of intracellular free calcium concentration [Ca2+]i. Changes in [Ca2+]i are involved in several events of differentiation and the embryogenesis process [16, 17]. Spitzer et al. [18] showed that naturally occurring patterns of Ca2+ transients encoded neuronal differentiation. Distinct frequency patterns of [Ca2+]i elevations were sufficient to promote neuronal differentiation, including physiological neurotransmitter receptors expression [19]. ATP and UTP are the main purinergic agonists activating P2X or P2Y receptors. These nucleotides can be rapidly degraded in the extracellular space by ectoenzymes to ADP or UDP, subsequently activating distinct P2Y receptors, or be finally degraded to adenosine, which is known to induce physiological responses via activation of P1 G protein-coupled receptors [20] (Fig. 1).
Fig. 1Purine-induced signaling pathway involves the activation of P1 adenosine and P2 purinergic receptors and purine hydrolysis by ectonucleotidases. The scheme demonstrates purinergic receptor activity present in glia-glia, neuron-glia, and neuron-neuron interaction during neurogenesis as well as in the metabolism of the adult brain
In this review article, we shall discuss the roles of purinergic signaling in neurogenesis such as cell cycle control during neural progenitor proliferation and differentiation as well as in maintaining physiology of neurons and glial cells and the involvement of purinergic receptors in pathophysiology. In addition, we shall outline state-of-the-art approaches used in investigation of P2 receptor function in physiological processes such as the use of antisense oligonucleotides, generation of knockout animals, and identification of new purinergic receptor subtype-selective drugs.
Study of purinergic receptor function during in vitro differentiation
During the development of the mammalian nervous system, neural stem cells and their derivative progenitor cells generate neurons by asymmetric and symmetric divisions [21]. P2 receptors were shown to be one of the first functionally active membrane receptors in chick embryo cells during gastrulation, in which ATP caused rapid accumulation of inositol triphosphate and Ca2+ mobilization in a similar way as acetylcholine (Ach) did via activation of muscarinic acetylcholine receptors, whereas other endocrine-acting substances such as insulin and noradrenaline (NA) induced much weaker effects in terms of intracellular calcium signaling [22, 23]. The induction of transient fluctuation in [Ca2+]i also denominated as calcium wave signaling allows for a coupling of spatial and temporal information. Thus, calcium waves have been proposed to play a role in mapping of neuronal networks [24] and to modulate neurogenesis during embryonic cortical development [25].
Neurotransmitters are prominent candidates for transcellular signals that could influence the development of embryonic neurons as they surround neural cells throughout brain development [26–29]. In addition, functional ligand-gated ionic channel receptors have been identified in neural progenitor cells prior to establishing cortical and subcortical synapses [30, 31]. In this context, the extracellular signaling mechanisms controlling the various transition steps involved in adult neurogenesis are still poorly understood. One approach used to identify the function of P2 receptors during development and differentiation is the use of in vitro models for neuronal and glial differentiation such as embryonic and adult neural progenitor cells (NPC), also known as neural stem cells (NSC), embryonic stem (ES), and embryonal carcinoma (EC) cells.
ES cells are obtained from the inner mass cell of the blastocyst. The differentiation of these cells closely resembles the in vivo process and, therefore, provides stable models for embryonic growth and development [32, 33]. ATP promotes cell proliferation acting through P2X3, P2X4, P2Y1, and P2Y2 receptors in murine ES cells [34]. Tissue-nonspecific alkaline phosphatase (TNAP) was also detected in these cells and used as a marker for their undifferentiated stage [35].
The neuronal differentiation of EC cells, originated from irradiated embryo cells [36], also resembles early neuronal development in vivo. P19 mouse EC cells express stem cell-specific marker proteins and their phenotypic changes in specific differentiation stages are similar to those of stem cells [37]. Recently, our laboratory [38] has determined gene and protein expression of P2 receptor subtypes throughout in vitro neuronal differentiation of P19 cells as well as in the undifferentiated cell stage suggesting the participation of purinergic signaling in initiating and directing differentiation. Differential expression and activity of P2Y1, P2Y2, P2Y4, P2X2 subtypes and P2X6 subunits were reported during neuronal maturation of P19 cells [38, 39]. As direct evidence for participation of purinergic receptors in neuronal differentiation, the presence of the antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), reactive blue 2, or suramin during differentiation of P19 neural progenitor cells (NPC) to P19 neurons resulted in reduced activity of cholinergic and glutamate NMDA receptors in differentiated P19 cells, pointing at a participation of P2Y1, P2Y2, and P2X2 receptors.
Other in vitro neuronal and glial differentiation models used to understand the purinergic signaling are neural stem cells or progenitor cells which are isolated from the subventricular region (SVZ) located in the lateral ventricles (type B cells) or in the subgranular region of the gyrus dentatus of the hippocampus (residual radial glia) or even from the subcortical parenchyma of the cerebral cortex of embryonic and adult brain [40–42]. These regions in the adult brain act as neural stem cell reservoirs. These cells are already advanced in their differentiation stage when compared to ES or EC cells. Since NSC and NPC are capable of differentiating in both functional neurons and glial cells, they possess potential therapeutic applications such as ES cells in regeneration therapy following neuronal loss.
These NPC differentiate into olfactory, cerebellar, and retinal neurons [40] in the presence of growth factors, neurotransmitters, vasoactive peptides in vivo [43], and growth factors such as epidermal growth factor (EGF), fibroblast growth factor 2 (FGF-2), and leukemia inhibitory factor (LIF) in vitro. When exposed to a high concentration of FGF-2 in suspension, proliferating NPC form tridimensional cell aggregates denominated as neurospheres, which following induction to differentiation express neuronal marker proteins such as β-III-tubulin, microtubule-associated protein-2 (MAP-2), and synaptophysin [44] and express P2X3 and P2X7 receptors which may contribute to early [Ca2+]i transients as prerequisites for further differentiation [41]. Shukla et al. [45] identified functional P2 receptors in adult mouse hippocampal progenitors in situ and the nucleoside triphosphate-hydrolyzing ectoenzyme (NTPDase) in type B cells of the SVZ [46] and in hippocampal progenitor cells. In adult murine NPC of SVZ, P2Y1 receptor activity mainly contributes to [Ca2+]i transients with some participation of P2Y2 receptors. The presence of the specific P2Y1 receptor antagonist MRS 2179 resulted in diminished cell proliferation in neurospheres due to reductions of [Ca2+]i transients. Similar results were obtained with NPC from SVZ of P2Y1 receptor knockout mice [47]. P2Y1 receptor-deficient mice are viable; however, they have deficits in platelet aggregation [48]. It is suggested that the purine signaling underlies autocrine or paracrine mechanisms and P2Y1 and P2Y2 receptors are important for NPC differentiation [47]. These models are useful tools to study the roles of P2 receptor signaling in early stages of development and differentiation. The importance of ATP release and purinergic signaling has not only been demonstrated in developmental progenitor cell expansion and neurogenesis, but also in persistent progenitor cells of the adult brain [49].
Expression of purinergic receptors during development of the central nervous system
Purinergic signaling pathways are also involved in embryonic neurogenesis in much the same way as already discussed for in vitro differentiation models. ATP mediates elevation of [Ca2+]i and proliferation of immortalized human stem cells from embryonic telencephalon and mouse embryonic neurospheres [50, 51]. Ca2+ waves through radial glial cells in slices of the embryonic rat ventricular zone are mediated by P2Y1 receptors. Disrupting Ca2+ waves between embryonic NPC reduced ventricular zone cell proliferation during the peak of embryonic neurogenesis [25].
ATP directly contributes to modulate network-driven giant depolarizing potentials in the rat hippocampus during early stages of postnatal development [52]. In the developing hippocampal system a trophic role of ATP and the involvement of P2 receptor subtypes in shaping interneuronal connections during neuronal differentiation have been suggested [53]. Alterations of the regulation of embryonic growth by purinergic receptors might be involved in the onset of morphological malformations [54]. During rat postnatal development ectonucleotidase activity in the cerebral cortex steadily increases, reaching maximum values at 21 days of age [55]. Several P2Y and P2X receptors were shown to be dynamically expressed in the pre- and postnatal central and peripheral nervous system [56–59]. ATP inhibited motor axon outgrowth during early embryonic neurogenesis, most likely through the P2X3 receptor, and it was speculated that P2X7 receptors might be involved in programmed cell death during embryogenesis [58].
From all of the studied P2X receptors, homomeric P2X2 receptors were the first expressed in the rat central nervous system (CNS) on embryonic day 14 (E14) [56]. On E14, heteromeric receptors were formed by P2X2/3 receptor subunits. P2X3 receptor immunoreactivity was detected in cranial motor neurons as early as on E11, when neurons exited the cell cycle and started axon outgrowth, as well as postnatally on days 7 and 14 (P7 and P14) [56, 60]. Moreover, expression of P2X3-containing heteromeric receptors and other subunits was developmentally regulated in nucleus ambiguous motoneurons [61]. From E14 onwards P2X7 receptors were also expressed in the embryonic brain. For instance, in primary cultures of human fetal astrocytes basal levels of P2X7 receptor mRNA transcription and protein expression were detected [62]. Sperlágh et al. [63] have demonstrated that ATP regulates glutamate release via activation of P2X7 receptors. P2X7 receptor-induced excessive glutamate release alters Ca2+ homeostasis, subsequently resulting in activation of the apoptosis-related caspase cascade [64].
P2X receptor expression was downregulated in Purkinje cells and deep cerebellar nuclei at P21 and P66 rat embryonic stages, with the exception of P2X5 receptors whose immunoreactivity in granular cells was increased [65]. Evidence for participation of P2X receptors in different developmental processes such as neurite outgrowth (involving P2X3 receptors), postnatal neurogenesis (related to P2X4 and P2X5 receptor expression), and cell death (possibly involving P2X7 receptors) was collected. However, P2X1 and P2X6 receptor subunits may not play a role in neuronal development [58].
Neocortical neurons from 2-week-old rats possess a quite elaborated purine-triggered signaling system which includes both P2Y and P2X receptor activation [66]. Weissman et al. [25] showed that [Ca2+]i waves and subsequent ATP release, with consequent P2Y1 receptor activation, accompanied radial glial cell-derived neurogenesis in cultured slices of the developing rat forebrain, as mentioned above. Moreover, the importance of calcium signaling for differentiation of NPC has been studied [67, 68], and direct evidence for the participation of P2Y1 receptor-activated pathways in the early development has been provided by Scemes et al. [69]. P2Y receptors (particularly the P2Y1 subtype) were widely expressed in the embryonic rat brain as early as on E11 [57]. There was a marked decrease in the concentration of mRNA coding for P2Y1 receptors and upregulation of mRNA transcription coding for P2Y2 receptors in freshly isolated astrocytes of developing rat hippocampus [57].
Functional interactions between neurons and glia: a physiological overview
An increasing amount of evidence, initiated by the neuron-glia unit idea proposed by Hyden [70], indicates that glial cells, once referred to as a simple support portion in the CNS, are now considered indispensable functional partners of neurons [71], both in physiological and pathological conditions. However, many questions remain unanswered: (1) how glia detects and interacts with neural function; (2) does neuron-glia signaling play a significant role in synaptic transmission and plasticity; and (3) how glial cells can communicate with other glial cells.
Another important subject related to the interaction between glia and neurons emerges in neurogenesis. There is now a general agreement that the adult mammalian nervous system possesses many characteristics of astrocytes. The importance of glia in neuronal development was confirmed in a recent study showing that the number of GFAP (glial fibrillary acidic protein)-containing cells was reduced following transgenic targeting of adult mouse subependymal and subgranular zones, resulting in an almost complete loss of neurogenesis [72, 73]. In addition to assisting migration of neurons to their correct position and managing neurite outgrowth to their final communication targets [74, 75], glial cells have become an essential key for understanding neuronal differentiation by promoting initial stem cell proliferation and instructing undifferentiated cells to adopt a neuronal fate [76, 77].
In the mature brain, the proximity of astrocytes to neuronal synapses or to the blood-brain barrier makes these cells appropriate to control water diffusion and ion concentration in extracellular spaces [71, 78]. In particular, astrocytes regulate homeostatic environment and neurotransmitter levels by functional syncytium, in which gap junctions and specific membrane carriers play an important role [79–81]. In addition, glial cells produce and release a vast number of neurotrophins, including fibroblast growth factor, nerve growth factor, and transforming growth factor, which directly interfere in neuron physiology and coordinate developmental processes [71, 82–85].
ATP release and degradation, connecting adenosinergic and purinergic systems
As already mentioned, it is well documented that glial cells may directly alter neuronal activity by releasing neurotrophins and consequently modulating neurotransmitter release in the synapse [86, 87]. One of the main mechanisms connecting the neuron-glia system is believed to be mediated by the release of glutamate from glial cells [88, 89]. In this context, growing evidence indicates that purinergic receptor ligands are widely involved in the cell-cell signaling mechanism by acting as neurotransmitters or neuromodulators released by glial cells to control synaptic transmission in the CNS, as part of multiple functions of astrocytes [22, 90, 91] (Fig. 1).
ATP is an ideal molecule for cell signaling due to its intrinsic properties such as its small size, diffusing molecule rate, instability and low concentration in the extracellular environment, and impossibility to cross the plasma membrane [92, 93]. These properties imply the presence of particular pathways for ATP release that could be associated with cellular excitation/response and cell-cell signaling [94, 95]. First, ATP may be stored in synaptic vesicles alone or with other neurotransmitters and then released, as a classic synaptic mechanism in the peripheral or central nervous system [96, 97]. Second, a nonvesicular mechanism of ATP release could be observed through gap junction hemichannels, ATP-binding cassette proteins, P2X7 receptor pores in glial cells, and via chloride channels [98–101]. Third, ATP could be released due to cytolysis or cell damage. While this is not a physiological mechanism, it takes place following biological trauma and contributes to pathological conditions [102].
Subsequent to these mechanisms, the metabolism of the released ATP is regulated by a vast number of different families of ectonucleotidases in the synaptic cleft, including the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and the ectonucleotide pyrophosphatase phosphodiesterase (E-NPP) which catalyze the degradation of ATP to ADP or AMP. The degradation to adenosine is mediated by ecto-5′-nucleotidase (E-5′-NT) and alkaline phosphatase [91, 103] (Fig. 1). Consequently, the reaction products resulting from the ATP hydrolysis may bind to P2 receptors, in the case of ADP, or to P1 receptors in the case of adenosine [104].
The adenosinergic receptor ligand adenosine is recognized as an important regulator of cellular homeostasis in the CNS and may be involved in the prevention or induction of apoptosis [105]. The reduction of ectonucleotidase activity in certain pathological conditions provided additional evidence for the accumulation of ATP in the extracellular environment [20]. Therefore, the complexity of the communication of neural and nonneural cells expands the functional significance by the interaction of the purinergic receptors in association with a variety of neurotransmitter systems.
ATP-mediated neuron-glia signaling
Novel studies in the purinergic field began to converge with glial research as it became more widely accepted that ATP is released through synaptic vesicles and thus accessible to perisynaptic glial cells, allowing them to detect neuronal activity. In particular, glial cells are responsive to ATP, as all types of glia, such as astrocytes, oligodendrocytes, microglia, and Schwann cells, express purinergic receptors [91]. In Schwann cells and oligodendrocytes, ATP-mediated signaling predominantly occurs through P2Y receptors, which in turn trigger intracellular Ca2+ release [106, 107]. However, the function of P2X1–6 receptors in astrocytes remains unclear, although P2X-mediated currents could be detected in astrocyte cells in culture, and P2X7 receptors are widespread in these cells with possible contribution to pathological conditions [108, 109].
Glial cells express many types of neurotransmitter receptors and conventionally are considered to be nonexcitable [110, 111]. However, a surprising observation was reported by Dani et al. [112] that synaptic transmission may propagate to glial cells as calcium waves, inducing membrane depolarization and regulating neurotransmitter release. These properties of glial cells suggest possible rapid communications between neurons and glia during synaptic transmission. This glial communication mechanism allows the released ATP to act onto adjacent astrocytes and neurons, thus supporting the propagation of Ca2+ waves in glial syncytium [113]. For example, in neuronal-glial cocultures prepared from hippocampus, ATP secreted by astrocytes was shown to inhibit glutamatergic synapses through activation of P2Y receptors [114].
The glial communication mechanism based on Ca2+ wave propagation could be inhibited by P2 receptor blockers or enzymes that rapidly hydrolyze extracellular ATP [115]. A stimulation applied to a single astrocyte in cocultures of rat forebrain astrocytes and associated neurons caused an elevation of [Ca2+]i and induced Ca2+ wave propagation in dorsal spinal cord through P2Y1 receptor activation [116] and glutamate release [117]. This new finding provided a parallel mechanism of intercellular communication that could allow astrocytes to detect synaptic function, propagate the information through neighboring glial cells, and then influence synaptic function in a distant part of the nervous system.
Purinergic receptor-calcium signaling in glial cells plays important roles during CNS development. P2X1, P2X4, and P2X7 receptors were expressed in microglia at rat embryonic stage (E16) [59]. Moreover, changes in P2X4 receptor expression in microglial cells during postnatal development of the rat cerebellum have been reported. P2X5 receptor immunoreactivity was also upregulated in microglia and granular cells. Both P1 and P2 receptors contribute to the modulation of oligodendrocyte (OP) development, since they have been shown to exert similar effects on OP proliferation and differentiation [118].
The majority of the studies of ATP action have been concerned with the short-term P2 receptor signaling that occurs in neurotransmission and in secretion [119]. Furthermore, there is increasing evidence that purines and pyrimidines can have trophic roles in neuritogenesis [120, 121], regeneration [122], and proliferation [123]. However, some purines by themselves have limited trophic effects in a few types of cells; they appear to be much more effective as neuritogenic agents when they are combined with other trophic factors, such as NGF. For instance, inosine and 5′AMP alone do not elicit neurite extensions in PC12 cells [124].
Heine et al. [53] demonstrated that P2 receptor activation induced fiber outgrowth in organotypic cocultures in rat hippocampus. Fiber outgrowth was inhibited in the presence of the purinergic antagonist PPADS, suggesting the involvement of P2 receptors. In another study, the synergistic interaction between bFGF and ATP was reported on DNA synthesis in primary cultures of rat cortical astrocytes. ATP and bFGF induced a twofold and tenfold incorporation of [3H]thymidine into astrocytes, respectively, but when ATP and bFGF were added at the same time a 50-fold increase in [3H]thymidine incorporation was observed [12].
Neuroprotection
ATP can activate P2X7 receptors in astrocytes to release glutamate, GABA, and also ATP which might regulate the excitability of neurons in certain pathological conditions [125]. It has been suggested that astrocytes can sense the severity of damage in the CNS by the amount of ATP released from damaged cells and that extracellular ATP concentration and the corresponding subtype of activated astrocytic P2 receptor modulate the tumor necrosis factor-α (TNF-α)-mediated inflammatory response [126]. After mechanical brain injury, the administration of PPADS facilitated the recovery of pathologically changed electroencephalograms [127]. These results suggest that interference with the ATP-induced excitatory responses could provide neuroprotection and possible therapeutic consequences.
Evidence for a neuroprotective role was also found for the adenosine A1 receptor in hippocampus. This cerebral region is highly sensitive to hypoxia and ischemia. The study of the action of hypoxia on synaptic transmission in hippocampal slices has suggested that substances being released during hypoxia, such as GABA, ACh, and even glutamate, may also play neuroprotective roles. However, the actions of these neurotransmitters become evident only when activation of P1 receptors is impaired, suggesting a critical role for this receptor during hypoxic events. These substances can operate in a redundant or even overprotective manner, acting as a substitute for some adenosine actions when the nucleoside is not operative [128].
Neuroimmune interactions
Microglia, the immune cells of the CNS, can be activated by purines and pyrimidines to release inflammatory cytokines such as IL-1, IL-6, and TNF-α. However, hyperstimulation of the immune reaction in the brain may accelerate neuronal damage. The P2X7 receptor is considered to have a potentially pivotal role in the regulation of various inflammatory conditions. ATP selectively suppresses the synthesis of the inflammatory protein microglial response factor through calcium influx via P2X7 receptors in microglia [129], which also leads to enhancement of interferon-γ (IFN-γ)-induced type II nitric oxide synthase (NOS) activity [130, 131]. P2X7 receptor activity also participated in ATP-induced IL-1 release from macrophages and microglia that had been primed with substances such as bacterial endotoxin [132] and was shown to stimulate the transcription of nuclear factor κB, TNF-α [133], the stress-activated protein kinases (SAPK)/JNK pathway [134], and the production of 2-arachidonoylglycerol, which is also involved in inflammation induction by microglial cells.
P2Y rather than P2X7 receptors seem to have a major role in the IL-6 production by microglial cells [135]. ATP evoked the release of plasminogen [136] and IL-6 [135]. The stimulation of microglia by either ATP or BzATP revealed neurotoxic properties and the involvement of the P2X7 receptor has been reported in excitotoxic/necrotic and apoptotic degeneration [109].
Neurological disorders
Epilepsy Several anti-epileptic agents reduce the ability of astrocytes to transmit Ca2+ waves, raising the possibility that blockade of ATP-induced [Ca2+]i transients in astrocytes by purinergic receptor antagonists could offer new treatments for epileptic disorders. Antiepileptic effects of adenosine are mostly due to the well-known inhibitory actions of P1 receptors on synaptic transmission in the hippocampus. However, as recently pointed out, adenosine actions are not limited to presynaptic actions on glutamate release [137]. The intraventricular injection of high doses of ATP in rats evoked severe chronic-tonic convulsions, whereas lower doses of ATP or adenosine elicited a kinetic state with muscle weakness [138]. P2X2 and P2X4 receptor expression in the hippocampus of seizure-prone gerbils was significantly reduced compared with that of normal gerbils [139]. GABAA receptors mediated modulation of expression of both P2X2 and P2X4 receptors, which may play an important role in the regulation of seizure activity in the gerbil hippocampus [139]. P2X7 receptors are thought to play a definite, but not yet well defined role in epilepsy. Treatment with the GABAB receptor agonist baclofen and antagonist phaclofen resulted in increased and decreased P2X7 receptor expression in hippocampus, respectively [140]. These purinergic receptor responses were interpreted as compensatory responses to the modulation of GABAB receptor function [140]. It is noteworthy to mention that this positive relationship between P2X and GABAA receptors was also reported for the spinal cord [141] and dorsal root ganglia (DRG) [142]. In these populations of neurons, ATP-mediated P2X receptor function may participate in neuronal transmission accompanied by GABA-mediated actions [139].
Pain The heteromeric channel comprised of P2X2 and P2X3 subunits was expressed almost exclusively in a subset of primary afferents implicated in nociception [143–145]. It has been observed that mechanical allodynia is reduced in mice with deleted P2X3 receptor genes [146, 147] in agreement with data obtained in rats that have been treated with intrathecal antisense oligonucleotides reducing expression of P2X3 receptors [148] or with the selective antagonist for P2X3 and P2X2/3 receptors A-317491 [148, 149]. P2X3 receptor knockout mice showed additional defects in afferent pathways.The P2X4 receptor is also implicated in pain sensation. Activation of dorsal horn microglia and tactile allodynia developing several days after ligation of a spinal nerve were greatly reduced when gene expression of P2X4 receptor in the dorsal horn had been inhibited by the presence of intrathecal antisense oligonucleotides [150]. Accordingly, intraspinal administration of microglia following induction of expression and activity of P2X4 receptors produced tactile allodynia in naive rats. Intrathecal administration of cultured brain microglia produced allodynia, but only when the cells had been pretreated with ATP [150]. The inhibition of P2X4 receptor activity in microglia might be a new therapeutic strategy for pain induced by nerve injury.
Alzheimer’s disease Alzheimer’s disease (AD) is caused by extracellular deposition of amyloid β-peptide, which can damage neurons, leading to their dysfunction and death [151]. ATP and, in particular, aluminum-ATP promoted the formation of thioflavin T-reactive fibrils of β-amyloid and an unrelated amyloidogenic peptide, which could be blocked by suramin [152].Microglial cells are believed to contribute to the progression of AD and are known to release proinflammatory neurotoxic substances. Extracellular ATP, acting through the P2X7 receptor, can alter β-amyloid peptide-induced cytokine secretion from human macrophages and microglia and thus may be an important modulator of neuroinflammation in AD [153]. P2X7 receptors mediate superoxide production in primary microglia, and the expression of this receptor subtype was specifically upregulated around β-amyloid plaques in a transgenic mouse model of AD [154].In contrast to the control human brain, the P2Y1 receptor was colocalized with a number of characteristic AD structures such as neurofibrillary tangles, neuritic plaques, and neuropil threads in the hippocampus and cortex [155]. In general, control brain tissue exhibited a greater and more abundant level of P2Y1 receptor immunostaining than AD tissue did, probably due to severe neuronal cell degeneration in most AD brains. The intense P2Y1 receptor staining observed over pathological AD structures might imply that this receptor is involved either directly or indirectly in signaling events mediating neurodegeneration of pyramidal cells. Alternatively, P2Y1 receptors might have other diverse signaling roles, possibly involved in the production of intracellular tau deposits or might even serve to stabilize these tangle structures in some way [156].
Ischemia/hypoxia Under pathological conditions of hypoxia or ischemia, extracellular purine nucleotides leak from damaged cells and thereby may reach high concentrations in the extracellular space [157]. A direct participation of extracellular ATP and P2 receptors in ischemic stress has been reported in various cellular systems [157–160]. For example, P2X2 and P2X4 receptor expression in neurons and microglia, respectively, in the hippocampus of gerbils was upregulated following transient global ischemia [161]. Increased P2X7 receptor expression in astrocytes, microglia, and neurons appears to contribute to the mechanisms of cell death caused by in vivo and in vitro ischemia [162, 163]. Following induction of ischemia P2X7 receptor mRNA transcription and protein expression were elevated in cultured cerebellar granule neurons and organotypic hippocampal cultures [163]. Hence, the P2X7 receptor is apparently an important element in the mechanisms of cellular damage induced by hypoxia/ischemia. In many cell types, the activation of the P2X7 receptor led to rapid cytoskeletal rearrangements, such as membrane blebbing and cell lysis [164]. P2Y1 receptors are intensely expressed in Purkinje cells in deep layers of the cerebral cortex and in ischemia-sensitive areas of the hippocampus [165]. In conclusion, extensive evidence demonstrates a postischemic time- and region-dependent upregulation of P2X2,4,7 and P2Y1 receptor subtypes in neurons and glial cells and suggests a direct role of P2 receptors in the pathophysiology of cerebral ischemia in vitro and in vivo.
Trauma and axotomy P2 receptors are suggested to be involved in neuronal reactions after axotomy. Colocalization and temporal coactivation of purinergic and nitrergic markers support this idea, indicating possible interactions between these two systems [166]. Following peripheral nerve lesions, P2X3 receptor expression in DRG neurons was changed [167]. The increased expression of P2X3 receptor mRNA in intact neurons indicates a role of this subtype in the post-injury pathomechanism in primary sensory neurons [167]. After spinal cord injury, large regions of the peritraumatic zone were characterized by a sustained process of pathologically high ATP release [168]. Spinal cord neurons express P2X7 receptors, and exposure to ATP led to high-frequency spiking, irreversible increases in [Ca2+]i and cell death. The administration of P2 receptor antagonists (PPADS, oxATP) after acute impact injury significantly improved functional recovery and diminished cell death in the peritraumatic zone [168]. The involvement of P2X1 and P2X2 receptors in neuronal reactions after hemicerebellectomy was also described [169]. Furthermore, neuronal NOS and P2 receptors were colocalized and showed temporal coactivation after cerebellar lesions, indicating a close relationship between these two systems [166]. In addition, in this mixed model of differentiation and axotomy, the colocalization of ataxin-2 (ax-2, involved in resistance to degeneration phenomena, which may be lost after mutation)-immunopositive cells and P2X2 receptors was demonstrated in neurons, and post-lesional induction of P2X1 receptor and ax-2 immunoreactivity was reported as well [170]. In vivo treatment of P2Y2 receptor-expressing sciatic nerves with ATP-γS increased expression levels of the growth-associated protein 43 (GAP-43) as a marker for axonal growth in wild-type but not in P2Y2 −/− mice [171].Possible therapeutic manipulations to modulate astrocytic proliferation and to diminish glial scar formation in the adult brain and during development include the use of drugs known to interfere with nucleotide synthesis. Pekovic et al. [172] showed that treatment with the purine nucleoside analogue ribavirin (Virazole; 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) downregulates the process of reactive gliosis after sensory motor cortex lesion of the adult brain and facilitates re-establishing synaptic connections with the denervated cells at the lesion site. This may be a useful approach for improving neurological recovery from brain damage. The antiproliferative effect of ribavirin is due to the inhibition of de novo nucleic acid synthesis after depletion of GTP and dGTP pools with consequent impairment of specific transduction pathways.
ATP-induced effects on cell cycle progression
There is evidence showing that extracellular ATP enhances the expression of cell cycle regulating proteins [173, 174]. Progression of the cell cycle is highly controlled. Cyclins are synthesized and degraded in a synchronous way due to changing transcription or proteolysis rates, thereby directing the periods of the cellular cycle. Cyclins interact with cyclin-dependent kinases (cdks) resulting in activation of their kinase activity, phosphorylating their targets and themselves, and regulating the specific progression of the cell cycle through checkpoints [175].
Proliferation rates in mammalians are largely determined during the G1 phase of the cell cycle. The relevant proteins include three D-type cyclins (D1, D2, and D3) that, in different combinations, bind to and allosterically regulate one of two cdk subunits, cdk4 and cdk6, as well as the E-type cyclins (E1 and E2), which govern the activity of a single catalytic subunit, cdk2 [176]. Various combinations of D-type cyclins are expressed in different cell types, whereas cyclin E-cdk2 complexes are ubiquitously expressed [177].
Two families of cdk inhibitors regulate the activity of G1-type cyclins-cdks complexes: the Ink4 family (p16, p15, p18, and p19), which blocks the activity of cyclin D-cdk4-6 complexes, and the Cip/Kip family (p21, p27, and p57), which preferentially inhibits cyclin E-cdk2 complexes and also acts as a scaffold for the catalytically active cyclin D-cdk4-6 complexes. In addition to cyclins and cdks, mitogen-activated protein kinase (MAPK) is also believed to have a role in induction of cell proliferation. Therefore, cyclin D-dependent kinases may play a role in controlling the cell cycle of embryonic and maybe neural progenitor cells. In addition MAPK is also believed to have a role in induction of cell proliferation. Extracellular ATP induces Ca2+-dependent MAPK activation via stimulation of P2 receptors in neonatal rat astrocytes [178]. On the other hand, cell proliferation is associated with activation of diverse proteins. Positive regulators include cyclins and their partners with catalytic activity (cdks), which are essential for progression of the cells through each phase of the cell cycle and various cell cycle checkpoints [179, 180]. The regulation of cyclin D1 expression is also mediated by the Ras/ERK signaling pathways [181, 182]. Raf/MEK/ERK and PI3-K/Akt signaling pathways can act in synergy to promote the G1-S phase cell cycle progression in both normal and cancer cells [183, 184]. The promoter for cyclin D1 contains an AP-1 site, and the ectopic expression of either c-fos or c-jun induces cyclin D1 mRNA expression [185, 186]. In many cell types, phosphatidylinositol (PI)-3-kinase-dependent signaling pathways also regulate cyclin D1 expression [187]. It was also reported that the control of the cell cycle regulatory proteins was dependent on PI3-kinase and p44/42 MAPK pathways, indicating that extracellular ATP alone is sufficient to induce cell cycle progression beyond the G1 phase of the cell cycle. These findings also suggest that, once P2 receptors are activated, protein kinase C (PKC) transmit signals to the nucleus through one or more of the MAPK cascades, which may include Raf-1, MEK, and ERK, and stimulate transcription factors such as myc, max, fos, and jun. Moreover, MAPKs are upstream regulators of cdk2 and cdk4 expression. It has been reported that p44/42 MAPK phosphorylation is essential and sufficient for the increase in cdk2 [188, 189] and decrease in p27Kip1 expression [190, 191]. However, Delmas et al. [192] provided evidence that p44/p42 MAPK activation triggers p27Kip1 degradation independently from cdk2/cyclin E in NIH 3T3 cells. As described above, ATP regulation of the MAPK and cdk-cyclin complex has not been elucidated in other types of cells [193].
It is documented in the literature that purinergic receptor inhibitors interfere with the S phase of the cell cycle. Neurospheres treated with the purinergic receptor antagonists reactive blue 2 or suramin are mostly in S phase (5.7 ± 0.3% or 8.4 ± 2.3%) when compared to untreated control neurospheres with 16.4 ± 1.8% of the cells being in S phase. Moreover, neurosphere cultures treated with suramin or reactive blue 2 showed an increase in the expression of the tumor suppressor p27 as a strong regulator of cell division [49].
The discussed findings led to the suggestion that extracellular ATP plays an important physiological role during mammalian embryonic development by stimulating proliferation of ES cells, and therefore P2 receptor agonists and antagonists might provide novel and powerful tools for modulating embryonic cell functions. In conclusion, P2X and P2Y purinergic receptors can promote proliferation of ES cells as well as of progenitor cell types by a mechanism by that ATP induces increases in [Ca2+]i, leading to activation of PKC, PI3-kinase/Akt, p38, and p44/42 MAPK, followed by an alteration in the cdk-cyclin complex with p21 and p27, which are involved in stimulation of cell proliferation.
Pharmacological approaches
Most purinergic receptors do not have specific inhibitors. Therefore, P2 receptor agonists and antagonists acting on most of the purinergic receptor subtypes are widely used in experimental approaches to study biological functions of these receptors. Such approaches are feasible, since these compounds mostly have higher affinities to some P2 receptor subtypes than to other ones. As an example, we have used suramin, PPADS, and reactive blue 2 to study the participation of P2Y1, P2Y2, and P2X2 receptors in neuronal differentiation of P19 EC cells [38].
One possible approach towards a subtype-specific inhibitor would be based on results from P2 receptor structure determination. Using site-directed mutagenesis it has been possible to understand which amino acids are involved in ATP binding and to identify allosteric sites in purinergic receptors. The knowledge obtained on location and structural features of ligand and inhibitor binding sites is used in rational based drug design of selective purinergic subtype antagonists. Alternatively, combinatorial libraries formed by vast amounts of possible ligands can be employed for discovery of subtype-specific inhibitors.
A-317491 was identified as a specific inhibitor for P2X2/3 and P2X3 receptors. In the presence of A-317491 both thermal hyperalgesia and mechanical allodynia were attenuated after chronic nerve constriction injury in which P2X3 homomeric and P2X2/3 heteromeric receptor activities were involved. Although active in chronic pain models, A-317491 was ineffective in reducing nociception in animal models of acute postoperative pain and visceral pain indicating that P2X3 and P2X2/3 receptor activation may not be a major mediator of acute postoperative or visceral pain [149]. MRS 2179 (2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate) was discovered as a specific inhibitor of P2Y1 receptor activity [194]. This compound has an efficient antithrombotic action in which P2Y1 receptors are involved [195].
Based on structure design or combinatorial library approaches specific agonists or antagonists may be discovered for other purinergic receptor subtypes. For instance, the SELEX (systematic evolution of ligands by exponential enrichment) technique provides a particularly promising approach for the discovery of such compounds. This technique is based on the reiterative presentation of a partial random RNA or single-stranded DNA library to a protein preparation containing a particular purinergic receptor subtype. RNA or DNA molecules bound to a target site on the receptor are displaced from the receptor and eluted by addition of an excess concentration of an unspecific purinergic receptor antagonist and amplified by reverse transcription polymerase chain reaction (PCR) or PCR to restore the library used for the next in vitro selection cycle. Using this approach, it was possible to identify inhibitors specific for isoforms of a target protein [196]. Our group prepared membrane protein fractions of 1321N1 cells stably transfected with rat P2X2 receptors and coupled them onto an immobilized artificial membrane (IAM) as matrix for affinity chromatography. The equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2′3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using [α-32P]-ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, as already shown by Trujillo et al. [197]. Moreover, this chromatographic assay can be used in in vitro selection procedures for RNA aptamers binding to P2X2 receptors from a combinatorial SELEX RNA library [198]. The development of a subtype-specific P2X receptor antagonist by using the SELEX technique or another combinatorial library-based approach shall serve as proof of principle and encourage further works to obtain such specific antagonists for all P2 receptor subtypes as tools for elucidating their biological functions and for possible therapeutic applications.
Conclusion
P2 receptor function is involved in most physiological processes and participates in neurotransmission in the CNS. Results obtained with mouse ES and P19 EC and neural progenitor cells suggest an important role of purinergic signaling in early embryogenesis, especially in cell proliferation, migration, and differentiation, with different subtypes of receptors participating in these processes. Our understanding of the biological functions of specific P2 receptor subtypes during CNS development and in the adult brain has increased due to the availability of knockout animals and specific inhibition of gene expression or activity of purinergic receptor subtypes. The importance of P2 receptor signaling in neuroprotection, neuroimmunity, and guiding neuronal differentiation, especially in glial and microglial cells, has been related to purinergic receptor expression. Most importantly, specific agonists and antagonists for individual P2 receptor subtypes are both needed for studying their involvement in biological processes. The discovery of such selective compounds will elucidate yet unknown biological functions of P2 receptor subtypes as well as open new avenues for therapeutic approaches to disease states in which purinergic receptor activity is involved.

knockout animal
atp
neurotransmitter
neural stem cells
p19 embryonal carcinoma cells


CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age

Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is associated with the expression of the multidrug resistance (MDR) proteins P-glycoprotein, encoded by the MDR1/ABCB1 gene, multidrug resistant-related protein (MRP/ABCC1), the lung resistance-related protein (LRP), or major vault protein (MVP), and the breast cancer resistance protein (BCRP/ABCG2). The clinical value of MDR1, MRP1, LRP/MVP, and BCRP messenger RNA (mRNA) expression was prospectively studied in 154 newly diagnosed AML patients ≥60 years who were treated in a multicenter, randomized phase 3 trial. Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count (respectively, p < 0.05, p < 0.001, p < 0.001 and p < 0.001). Higher BCRP mRNA was associated with secondary AML (p < 0.05). MDR1 and BCRP mRNA were highly significantly associated (p < 0.001), as were MRP1 and LRP mRNA (p < 0.001) expression. Univariate regression analyses revealed that CD34 expression, increasing MDR1 mRNA as well as MDR1/BCRP coexpression, were associated with a lower complete response (CR) rate and with worse event-free survival and overall survival. When adjusted for other prognostic actors, only CD34-related MDR1/BCRP coexpression remained significantly associated with a lower CR rate (p = 0.03), thereby identifying a clinically resistant subgroup of elderly AML patients.
Introduction
Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is often associated with the expression of (membrane) transport-associated multidrug resistance (MDR) proteins. Expression of P-glycoprotein (P-gp), encoded by the MDR1 gene, is an independent adverse prognostic factor for response and survival in de novo AML [1–4]. Moreover, it has been shown that besides P-gp, also the MDR-related protein (MRP1/ABCC1) and the lung resistance-related protein (LRP), also designated as the major vault protein (MVP), are expressed in AML. However, the prognostic significance of the latter resistance proteins has not been settled [3, 5–7]. Some years ago, a new drug resistant protein, i.e., the breast cancer resistance protein (BCRP/ABCG2), which is the equivalent of the mitoxantrone (MXT) resistant protein and the placental ABC transporter (ABCP), was found to be expressed in AML [8–13]. The precise role of either resistance proteins among poor risk AML such as in patients of older age has not been established. This study prospectively investigated the relevance of MDR1, MRP1, LRP, and BCRP messenger RNA (mRNA) expression in combination with known prognostic characteristics like CD34 expression, white blood cell (WBC) count, and secondary AML as possible denominators of response and survival in patients with AML aged 60+ who were treated in the same clinical trial.
Patients and methods
Patients
A group of 154 patients with AML aged 60 years or older were included in the present study. All patients were enrolled between May 1997 and February 1999 in an international, multigroup, randomized phase 3 trial performed under auspices of the Dutch–Belgian Hemato-Oncology Cooperative Group and the UK Medical Research Council [14]. In that trial, 419 eligible white patients ≥60 years with previously untreated de novo and secondary AML (M0–M2 and M4–M7 according to the French–American–British [FAB] classification [15]) were randomized to receive two cycles of induction chemotherapy consisting of daunorubicin (DNR) and cytarabine (ara-C) with or without the P-gp inhibitor PSC-833 (Valspodar, Amdray®; Novartis Pharma, Basle, Switzerland). Patients in both arms in complete remission after these two cycles were to receive one consolidation consisting of ara-C, MXT, and etoposide. Inclusion criteria, clinical characteristics, treatment, and outcome of the phase 3 trial have been previously reported [14].
Bone marrow (BM) aspirates had been collected at diagnosis for the analysis of P-gp function and expression, as described previously [14]. Selection of patients for our study was based on availability of sufficient purified AML blast samples in our tissue bank, which was the case for 154 patients.
This study was approved by the ethics committees of the participating institutions and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients before randomization.
Methods
BM aspirates were obtained in heparinized tubes. Mononuclear BM cells were collected by Ficoll Hypaque density gradient centrifugation (density 1.077 g/m3; Pharmacia, Uppsala, Sweden). To obtain purified samples with more than 85% of blasts, T-cell depletion and adherence depletion was performed as previously described [16]. Cells were cryopreserved in Dulbecco modified Eagle medium (DMEM; Gibco, Paisley, UK) supplemented with 10% dimethyl sulfoxide (Merck, Darmstadt, Germany) and 20% fetal calf serum (FCS; Gibco) and stored in liquid nitrogen. On the day of the experiments, BM cells were thawed. Cells were washed and resuspended in DMEM supplemented with 10% FCS. Before RNA and DNA isolation, cells were washed with phosphate-buffered saline (Gibco).
MDR1, MRP1, LRP, and BCRP mRNA analysis
The drug resistance proteins were analyzed using the methods that we previously reported [11]. In brief, total RNA was isolated using the TRISOLV™ extraction as described by the manufacturer (Biotecx, Houston, TX). RNA was aliquoted and stored at −80°C. RNA samples were analyzed for RNA integrity by gel electrophoresis. cDNA was synthesized by the use of the TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA), diluted, aliquoted, and stored at −80°C. Quantitative RT-PCR was used to measure the mRNA expression levels of MDR1, MRP1, LRP, and BCRP by Taqman-chemistry on an ABI PRISM 7700 sequence detector (Applied Biosystems) using two endogenous reference genes, i.e., glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase.
Definition of endpoints
The clinical endpoints have been defined previously [14]. In brief, complete response (CR) was defined as a normocellular BM with <5% blasts, no Auer rods, and no evidence of extramedullary involvement. Because data on peripheral blood recovery within 60 days were not always available, they were not considered as a criterium for CR. Patients who relapsed or died within 28 days after CR were considered as not having achieved a CR. Event-free survival (EFS) was calculated from the date of randomization until no CR on induction therapy, relapse after CR, or death in CR, whichever came first. Patients who did not reach CR were considered failure for EFS at 1 day after randomization. Disease-free survival (DFS) was determined for all patients who achieved CR on induction therapy and was calculated from the date of CR until relapse or death, whichever came first. Overall survival (OS) was measured from randomization until death from any cause. Patients who were still alive at the date of last contact were then censored.
Statistical analysis
The original phase 3 trial had been designed to detect with a power of 80% an increase in 2-year EFS from 9.5% in the control arm (without PSC-833) to 18% in the PSC-833 arm (two-sided significance level α = 0.05) and included 419 eligible patients.
mRNA data were obtained from a subset of 154 patients with sufficient BM samples in our tissue bank available for analysis. Baseline parameters of interest were MDR1, MRP1, LRP, and BCRP mRNA expression. Clinical endpoints were CR rate, EFS, DFS, and OS.
Baseline characteristics of patients with or without mRNA expression data available were compared using the Fisher exact test or the Pearson χ2 test in case of discrete variables, whichever appropriate, or the Wilcoxon rank-sum test in case of continuous variables. The association between patient baseline characteristics and mRNA expression levels was analyzed using the Pearson χ2 test of the Spearman rank correlation test, whichever was appropriate. The prognostic value of mRNA levels with respect to CR rate was determined using logistic regression [17] whereas the impact of MDR1, MRP1, LRP, and BCRP on EFS, DFS, and OS was analyzed with Cox regression analysis [18]. For this purpose, the natural logarithm of the mRNA expression levels of the four resistance genes were included in the analyses because of the very skewed distribution of the original mRNA levels. In addition, the outcome of patients with coexpression of MDR1 and BCRP was evaluated to confirm the poor prognosis of AML with MDR1/BCRP coexpression reported by Benderra et al. [19] in patients with a median age of 45 years or older. These patients were defined as having mRNA levels of these two drug resistance genes equal to or higher than the median. Their outcome was compared to the other patients with at least one of the MDR1 and BCRP mRNA expression levels below the median. Logistic regression and Cox regression analyses were performed unadjusted, as well as adjusted for other prognostic factors, i.e., secondary AML, natural logarithm of WBC count, square root of percentage CD34+ cells, and cytogenetic risk (favorable/intermediate versus unfavorable versus unknown), as well as for treatment arm in the phase 3 trial, as about half of the patients had been randomized to receive PSC-833 in addition to their chemotherapy. Kaplan–Meier curves [20] were generated to illustrate survival and were compared using the log-rank test [21]. All reported p values are two-sided and, in view of the exploratory nature of these analyses, were calculated without adjustment for multiple testing. p values ≤ 0.05 were considered statistically significant.
Results
In the phase 3 trial, a total of 419 untreated patients with AML aged 60 years and older were randomized to receive two induction cycles with or without PSC-833. As reported, no difference was found between both treatment arms as regards CR rate (54% in the PSC-833 arm versus 48% in the control arm, p = 0.22), 5-year EFS (7 versus 8%, p = 0.53) nor DFS (13 versus 17%, p = 0.06) and OS (10% in both arms, p = 0.52) [14]. We previously reported the role of functional MDR1 expression with respect to clinical outcome in these patients.
In 154/419 of the patients, sufficient BM cells were available in our tissue bank to investigate the mRNA expression level of the drug resistance genes MDR1, MRP1, LRP, and BCRP. This subgroup consisted of a representative group according to age, gender, CD34 expression, cytogenetics, and FAB classification (Table 1). In this test group, a higher WBC count at diagnosis was observed than in the other 265 patients, and relatively more patients had been randomized to the PSC-833 arm (57 versus 45%, p = 0.02). There was no significant difference in the levels of MDR1, MRP1, LRP, nor BCRP mRNA expression between the two treatment arms (data not shown). The CR rate and survival endpoints were also similar in both patient groups (Table 1). However, patients with mRNA data in the PSC-833 arm had a higher CR rate (61 versus 40%, p = 0.02), whereas this was 54 versus 48% (p = 0.22) in all 419 patients.
Table 1Comparison between patients with or without data available for expression of the drug resistance genes Drug resistance genes evaluated Yes N (%)No N (%)Total N (%)pNumber of patients154265419Patient characteristicsMedian age, (range)67 (60–85)68 (58–85)67 (58–85)0.52Sex0.26 Male86 (56)163 (62)249 (59) Female68 (44)102 (38)170 (41)Secondary AML31 (20)73 (28)104 (25)0.09Median WBC count (×109/l; range)19.1 (0.1–389)5.6 (0.5–300)8.9 (0.1–389)0.001N146243389Median % CD34+, (range)32.5 (0.1–97.9)29.7 (0.1–93.7)30.3 (0.1–97.9)0.50N152157309Cytogenetic risk classificationa0.12 Favorable3 (3)2 (1)5 (2) Intermediate90 (80)132 (73)222 (76) Unfavorable19 (17)47 (26)66 (23) No data42 (n.i.)84 (n.i.)126 (n.i.)Treatment arm randomized0.02 DNR/ara-C66 (43)145 (55)211 (50) DNR/ara-C +PSC-83388 (57)120 (45)208 (50)Treatment outcomeCR rate, % (95% CI)52 (44–60)50 (44–56)51 (46–56)0.73EFS, % (95% CI)0.72 1 year23 (17–30)23 (18–28)23 (19–27) 5 years9 (5–14)7 (4–11)8 (5–11)DFS, % (95% CI)0.81 1 year38 (27–48)39 (31–48)39 (32–45) 5 years17 (10–26)14 (9–21)15 (11–21)OS, % (95% CI)0.31 1 year42 (34–50)41 (35–46)41 (36–46) 5 years14 (9–20)8 (5–12)10 (7–14)The results indicate that, apart from WBC count, there are no differences between the two subgroups.N number of patients with data (if not available for all patients); n.i. not included when calculating percentagesaClassification of cytogenetic abnormalities only for 293 patients with successful cytogenetics. Favorable risk: t(8;21), inv(16) or t(16;16). Unfavorable risk: the presence of monosomies or deletions of chromosomes 5 or 7, abnormalities of the long arm of chromosome 3(q21;q26), t(6;9), abnormalities involving the long arm of chromosome 11 (11q23), or complex cytogenetic abnormalities (defined as at least three unrelated cytogenetic abnormalities in one clone). Patients who did not meet the criteria for favorable or unfavorable risk were classified as intermediate risk [14].
The mRNA expression levels of the resistance genes were not significantly associated with the age of the patients (Table 2). MRP1 and LRP expression showed a strong positive correlation with WBC count. Negative associations of MDR1 and BCRP with WBC count were observed. A significant positive association was found between CD34 and MDR1 and also with BCRP mRNA expression. No significant correlation was found between MRP1 nor LRP, and CD34 expression (Table 2). Interestingly, secondary AML cases had a significantly higher expression of BCRP (p < 0.05) and lower MRP1 and LRP levels (both p < 0.01, Table 2). In the vast majority of our patients, also P-gp efflux and expression data were available. Function and expression data and MDR1 mRNA expression levels were highly correlated (p < 0.001), which was published recently [22].
Table 2Association between clinical patient characteristics and the mRNA expression of the four drug resistance genes and MDR1/BCRP coexpression mRNA expression ofMDR1MRP1LRPBCRPMDR1/BCRP coexpressionCharacteristic Age0.15−0.01−0.090.090.07(153)(153)(153)(137)(147) Secondary AML0.06−0.22**−0.21**0.19*0.12(153)(153)(153)(137)(147) WBC count−0.17*0.28***0.36***−0.36***−0.35***(145)(145)(145)(131)(139) CD34+0.54***0.14−0.080.17*0.27**(151)(151)(151)(135)(145) Unfavorable 0.11−0.05−0.23*0.130.10 Cytogenetic risk(111)(111)(111)(98)(106)Unfavorable cytogenetic risk was defined by the presence of monosomies or deletions of chromosomes 5 or 7, abnormalities of the long arm of chromosome 3(q21;q6), t(6;9), abnormalities involving the long arm of chromosome 11 (11q23), or complex cytogenetic abnormalities (defined as at least three unrelated cytogenetic abnormalities in one clone)Each cell displays the Spearman rank correlation coefficient between two variables and, between brackets, the number of patients with both variables available.*p < 0.05; **p < 0.01; ***p < 0.001
In this cohort of patients of higher age with AML, MDR1 and BCRP were highly associated (p < 0.001), just as were MRP1 and LRP mRNA (p < 0.001; Fig. 1). A negative association was found between BCRP and MRP1 and between BCRP and LRP (both p < 0.001; Fig. 1). The 40 patients with coexpression of BCRP and MDR1 had significantly higher CD34 expression (median 39.5% [range 0.1–97.7%] versus 25.9% [range 0.1–97.9%]; p = 0.001) and a lower WBC count (median 4.5 [range 0.8–300]×109/l versus 28.1 [range 0.1–389]×109/l; p < 0.001). No significant correlation of MDR1, BCRP, or coexpression of MDR1 and BCRP was found with unfavorable cytogenetics (p = 0.4; Table 2).
Fig. 1Association between MDR1, MRP1, LRP, and BCRP mRNA expression levels. Each dot represents the expression of two drug resistance genes in one patient. The Spearman rank correlation coefficient has been calculated, along with the corresponding p value. Both the x- and y-axis have a logarithmic scale trim(X)* indicates that the 2.5% smallest and largest values of X have been shrinked; r, Spearman rank correlation coefficient; and p, p value. The results show a significant positive correlation between MDR1 and BCRP mRNA expression as illustrated by the p value and correlation coefficient. In addition, MRP1 and LRP are highly associated. BCRP shows a negative correlation with MRP1 and LRP
To assess the clinical relevance of the four resistance genes, their expression was evaluated with regard to CR rate and survival data, respectively. The median follow-up of the 25 patients still alive was 58 months (range, 1–80 months). Univariate logistic regression analysis showed that higher MDR1 mRNA expression predicted for a lower CR rate (log[MDR1]: odds ratio [OR]=0.75, 95% confidence interval [CI] 0.61–0.93, p = 0.009), whereas MRP1, LRP, and BCRP mRNA were not associated with CR (Fig. 1). MDR1 expression was also associated with a worse EFS (log[MDR1]: hazard ratio [HR]=1.14, 95% CI 1.03–1.27, p = 0.01) and OS (log[MDR1]: HR = 1.16, 95% CI 1.05–1.29, p = 0.004). Similar results were also obtained for MDR1/BCRP coexpression (Table 3; Fig. 2). When the analyses were performed with adjustment for other prognostic factors, as described in the “Statistical analysis”, only MDR1/BCRP mRNA coexpression remained significantly associated with a lower CR rate (OR = 0.37, 95% CI 0.15–0.91, p = 0.03), whereas a trend was observed for worse EFS (Table 3). On the other hand, higher CD34 expression was significantly associated with a lower CR rate (square root[CD34]: OR = 0.86, 95% CI 0.76–0.98, p = 0.02) and with worse EFS (HR = 1.12, 95% CI 1.06–1.19, p < 0.001), DFS (HR = 1.19, 95% CI 1.09–1.30, p < 0.001), and OS (HR = 1.17, 95% CI = 1.10–1.25, p < 0.001).
Table 3Prognostic value of drug resistance gene expression w.r.t. CR rate, EFS, DFS from CR, and OS CR rateEFSDFSOSOR95% CIpHR95% CIpHR95% CIpHR95% CIpMDR1 Univariate0.750.61–0.930.0091.141.03–1.270.011.130.97–1.300.111.161.05–1.290.004 Adjusted0.770.58–1.030.081.050.91–1.210.480.950.77–1.180.671.000.87–1.160.97MRP1 Univariate1.060.83–1.350.631.020.90–1.150.791.070.89–1.290.471.110.97–1.260.12 Adjusted1.220.90–1.660.201.000.87–1.150.981.120.92–1.370.261.050.91–1.210.54LRP Univariate1.160.94–1.430.160.950.86–1.060.360.980.84–1.140.790.970.87–1.080.60 Adjusted1.220.93–1.610.150.990.87–1.120.831.060.89–1.270.520.980.86–1.120.78BCRP Univariate0.840.66–1.060.141.040.91–1.180.580.950.77–1.160.600.960.84–1.100.58 Adjusted0.790.59–1.06 0.120.990.86–1.140.920.840.66–1.060.140.900.77–1.050.19MDR1/BCRP co-expression Univariate0.380.18–0.800.011.631.11–2.370.011.650.90–3.010.111.471.00–2.160.05 Adjusted0.370.15–0.92 0.031.530.98–2.380.061.370.67–2.820.391.160.74–1.830.51Results of logistic (for CR rate) and Cox regression (for survival) analyses, either univariate (=unadjusted) or adjusted for treatment arm, secondary AML, WBC count (natural logarithm), % CD34+ (square root), and cytogenetic risk (favorable/intermediate versus unfavorable versus unknown), are shown for each of the four drug resistance genes MDR1, MRP1, LRP, and BCRP (natural logarithm of mRNA expression levels) and for MDR1/BCRP co-expression.Fig. 2Survival of elderly AML patients with and without coexpression of MDR1 and BCRP mRNA. a Event-free survival, b disease-free survival, c overall survival. pos indicates patients with coexpression of MDR1 and BCRP; and other, patients without coexpression
Discussion
This is the first comprehensive analysis of the effect of the major classical MDR genes in a cohort of elderly patients with AML homogeneously treated in a prospective clinical trial [14]. A wide range of expression of the various resistance genes was observed, consistent with previous studies and with comparable median values [9–11, 23]. Our results show that MRP1, LRP, and BCRP are not associated with CR rate or survival endpoints in patients with AML aged 60 years or older, indicating that the clinical relevance of the expression of these genes is limited in this patient population. This study confirms previous reports, which showed the unique prognostic role of MDR1 expression—which was however highly correlated with CD34 expression—in drug resistance in elderly AML (Table 3), in contrast to the prognostic value of MRP1 expression in AML, which has shown conflicting results, whereas currently, LRP is no longer thought to be important for clinical drug resistance [4, 5, 7, 24–27]. Recently, two studies in, respectively, 40 and 31 adult AML patients showed no effect of BCRP gene expression on CR rate, whereas OS was lower in patients with the highest BCRP expression [10, 23]. Damiani et al. [28] showed that BCRP expression did not influence achievement of complete remission in AML patients with a median age of 53 years and normal karyotype, however, BCRP expression was associated with higher relapse rate. In 59 children with de novo AML, a higher BCRP expression was observed in patients who did not reach CR, but this was not translated in poorer survival [29]. Benderra et al. [19] indicated that BCRP gene expression was an adverse prognostic factor for CR in a group of 149 relatively younger adult AML patients but only in patients treated with DNR and MXT and not with idarubicin. In our cohort of elderly AML patients who were all treated with DNR, whereas MXT was given as consolidation therapy after reaching CR, a significant correlation of BCRP mRNA expression with lower CR rate could not be shown.
Our study confirms that BCRP and MDR1 are coexpressed in AML patients with higher age as has been suggested previously from studies in smaller groups of relatively younger AML patients [9–11, 28]. Until now, only two studies have evaluated the clinical value of coexpression of MDR1 and BCRP in a sufficient number, although relatively younger adult AML patients [19, 28]. Benderra et al. [19] showed that CR rate was only 45% in the patients with coexpression of BCRP and MDR1 (+/+) in comparison with 66% in the MDR1/BCRP−/+ and +/−group and 90% in the MDR1/BCRP−/−group (p = 0.003). Moreover, a significantly lower DFS and OS were found in the MDR1/BCRP+/+group. Damiani et al. [28] found a trend towards a higher relapse rate in the small group of BCRP+/MDR1+patients, indicating that this represents a robust resistant AML phenotype, consistent with our findings in elderly AML. The recent finding that BCRP and MDR1 expression was mainly found in the most resistant group of AML, using gene expression profiling, underscores the role of these drug resistance genes in AML [30].
However, this study shows, that the prominent prognostic role of CD34 expression in elderly AML should be emphasized, as higher CD34 expression was adversely associated with all clinical endpoints. MDR1 and BCRP but not MRP1 and LRP mRNA expression were found to be associated with high CD34 expression in these elderly AML patients, which may explain why MDR1 was no longer significant for CR rate, EFS, and OS when adjusted for other prognostic variables including CD34. In the past, MDR1 expression has been linked to the CD34-positive hematopoietic stem cell compartment of the leukemia subtype. In two other studies in younger AML patients, no overexpression (on mRNA and protein level) of BCRP in the CD34-positive blast population of clinical AML samples was found [13, 19]. In contrast, earlier studies in mice demonstrated high levels of BCRP and MDR1 expression in normal hematopoietic stem cells [31–34]. Previously, BCRP expression in subsets of stem cells has been reported, indicating that high BCRP expression may exist in CD34+/CD38− cells or in CD34+/CD33−cells [12, 35]. The differential expression of BCRP and MDR1 in specific subsets of hematopoietic stem cells is consistent with the side population phenotype as proposed by Goodell et al. [36] who claim that BCRP expression can be separated from those expressing the other ABC proteins. This would suggest that BCRP is expressed in even less differentiated hematopoietic stem cells than MDR1 [19]. In our study in AML, these immature subsets could not be separately investigated, however, the unique BCRP/MDR1+/+subgroup of patients reflects an immature leukemic cell type that has a very resistant phenotype in vivo, illustrated by a low CR rate and poor outcome (Table 3; Fig. 2).
This is the first study in which a correlation was found between secondary AML and a high expression of BCRP mRNA but not the other resistance proteins. In addition to our previous report that BCRP is frequently upregulated in patients with AML at relapse, we now demonstrate that expression of BCRP is representative of secondary AML, which is especially observed in elderly patients [11, 29]. Recently, Ross [37] suggested that MDR modifiers may be of benefit for patients with multiple dysplastic features. This may suggest that BCRP is upregulated in diseases in which exposure to xenobiotics during life plays an etiologic role.
We conclude that coexpression of CD34-related coexpression of MDR1 and BCRP reflects a clinically resistant subgroup of elderly AML. In this age group, only BCRP is correlated with secondary AML. As such, the development of new treatment strategies for elderly AML patients may focus on modulation of drug resistance targeting both BCRP and MDR1.