A key goal in hippocampal research is to understand how neuronal activity is generated and organized across hippocampal subregions to enable memory formation and retrieval. Neuronal activity in CA2 is regulated by spatial and social investigation as well as by novelty , and CA2 Using chemogenetics and electrophysiology, we show that CA2 contributes to slow γ oscillations in CA1 during novel animal investigation and fast γ during novel animal or object investigation in a layer-specific manner reflective of CA2 connectivity. Slow γ oscillations in CA1 rely on CA3 and CA2 input during running, and CA2 neuronal activity is required for social memory. Our findings of reduced slow γ oscillations in CA1 layers targeted by CA2 axons on reduction of CA2 activity support the involvement of CA2 in slow γ oscillations, and the finding that slow γ oscillations were impaired by CA2 silencing selectively during novel animal investigation are consistent with a role for CA2 in social cognition.The mammalian hippocampus is critical for the formation of long-term episodic memories . DespiteRodent hippocampal oscillations are dominated by three frequencies of activity: theta ∼6–9 Hz; , γ ∼25–, and ripAmigo2  to test whether CA2 contributes to γ oscillations in CA1 during novel object and social stimulation.Given the evidence for CA2’s role in social memory and that CA1 γ oscillations are regulated in a layer-specific manner reflective of its upstream inputs , 2016, wAmigo2-icreERT2 mice. Mice were housed under a 12/12 h light/dark cycle with ad libitum access to food and water. Mice were naive to any treatment, procedure or testing at the beginning of the experiments described here. Mice were group-housed until the time of virus infusion, at which point they were singly housed. All procedures were approved by the Institute’s Animal Care and Use Committee and were in accordance with the National Institutes of Health guidelines for care and use of animals.Experiments were conducted in adult male (more than eight-week-old) 12 genome copies/ml. For chemogenetic manipulation using hM4Di-mCherry, mice were bilaterally infused with 0.5 μl of AAV5-hSyn-DIO-hM4Di-mCherry into each hemisphere of the brain.All AAV viruses were produced by the Gene Therapy Center Vector Core at the University of North Carolina at Chapel Hill and had titers of >10Amigo2-icreERT2 mice began daily tamoxifen intraperitoneal injections (100 mg/kg tamoxifen dissolved in warmed corn oil) for a total of 7 d. Animals were allowed to recover from tamoxifen treatment for one week before undergoing electrode implantation to allow sufficient time for estrogen receptor translocation to the nucleus and subsequent recombination.For virus-infusion surgery, mice were anesthetized with ketamine  and xylazine , then placed in a stereotaxic apparatus while on a heated pad. Sedation was maintained with 1.5–2.5% isoflurane during surgery if animals regained reflex responses at any point during the surgery. Following three swabs with betadine and saline rinse, an incision was made down the midline of the scalp, a burr hole was drilled above each target region and viruses were microinjected using a 1-μl Neuros Hamilton syringe at a rate of 100 nl/min. Following infusion, the needle was left in place for five additional minutes to allow for diffusion of the virus before slowly being retracted. Injection coordinates for CA2 were −2.30 mm AP, ±2.50 mm ML, and −1.90 mm DV from bregma. The scalp was then sutured shut and animals were administered buprenorphine  for pain and returned to their cages. Two weeks following AAV infusion, in vivo electrophysiology were implanted with NeuroNexus A1x32 silicone electrodes (NeuroNexus). Mice were anesthetized with ketamine  and xylazine , then placed in a stereotaxic apparatus while on a heated pad. An incision was made in the scalp, and the skull was cleaned and dried. A screw for the electrode reference wire was positioned ∼4 mm posterior and 2 mm lateral to bregma over the right cortical hemisphere and a screw for the ground wire was positioned in the skull over the cerebellum. Three additional anchor screws were secured to the skull. Electrodes were then lowered into the hole drilled over dorsal CA1: −1.30 mm AP, −1.40 mm ML, −1.85 mm DV from bregma, and the reference and ground wires were attached to their respective screws. The NeuroNexus A1x32 electrode is designed with 32 recording sites arranged linearly along a single shank, spaced 25 μm apart, providing a tissue coverage range of 775 μm.At least one week after the last tamoxifen treatment, mice for Amigo2-icreERT2 line, naive to any experimental testing before being used in the test session. The novel object stimulus  or vehicle alternated across days, as did the type of stimulus presented . All recordings were made during the animal’s light phase under red light illumination to encourage exploration. For these experiments, animals were allowed to freely explore a clear Plexiglas cage (40.5 × 24.5 cm). Bedding was placed in the bottom of the cage (as in home cages) to encourage exploration. Curtains surrounded the cage to prevent viewing of the experimental room and limit light exposure. The recording protocol A proceedstimulus C consistNeural activity was transmitted via a 32-channel wireless 10× gain head stage  and was acquired using the Cerebus acquisition system (Blackrock Microsystems). Continuous LFP data were bandpass filtered at 0.3–500 Hz and stored at 1000 Hz. For behavioral tracking, videos of recording sessions were acquired with a sampling rate of 30 Hz using the Neuromotive Software (Blackrock Microsystems) and an infrared camera. Behavior was analyzed offline using the Ethovision XT (Noldus) behavioral tracking software. Subjects’ smoothed instantaneous velocity, measured from reflective tape on the headstage, was used to isolate periods of running from periods of relative immobility. Samples in which the animal’s smoothed instantaneous velocity exceeded a threshold of ≥7 cm/s were designated as periods of running and excluded from analysis. The 7-cm/s threshold was selected based on manual optimization of tracking parameters to best divide periods of quiet immobile rest from active traversing of the environment. For selection of periods in which subjects were actively investigating stimuli, a virtual 10-cm circle was drawn around the wire mesh cup in which stimuli were presented, and periods in which the subject’s body, nose, or tail were located within the circle were isolated B,C. For All neuronal data analyses were performed with MATLAB version R2016b (MathWorks) using custom written scripts for (1) selection of LFP samples corresponding to periods in which subjects performed behaviors of interest, (2) artifact rejection, and (3) spectral analysis of LFP samples using a family of Morlet wavelets. First, behavioral vectors were generated from tracking data generated by Noldus Ethovision XT to designate periods in which subjects were both immobile and inside the virtual circle drawn around the stimulus. The behavioral vectors were then used to match periods in which animals performed behaviors of interest to the appropriate LFP samples. Second, noise and muscle artifacts were removed from recordings based on a 6-standard deviation rejection threshold; when detected, a 3-s window around the noise threshold crossing was removed. Third, spectral analysis was performed using a family of 80-wavelet carrier frequencies which increased logarithmically and had a low-frequency cutoff of 0.5 Hz and a high-frequency cutoff of 100 Hz. For each recording session, the time-frequency representation of the data were obtained by convolving the LFP with the family of wavelets to produce a vector of the same length as the original LFP data.Over the eight recording days, each of the four-possible drug and stimulus combinations  were delivered twice, and the LFP power values from these two repeat sessions were averaged. For statistical analysis of LFP power we focused on the theta (6–9 Hz), slow γ (25–55 Hz), and fast (55–100 Hz) γ frequencies ranges. An average was calculated for each frequency band separately by averaging all wavelet values that were used to generate the time-frequency representation of that band. For example, the slow γ band was computed using 19 wavelets, so these 19 values were averaged to yield a single value for the slow γ band. These values were calculated for each hippocampal layer in each subject and used for group comparisons of power under the different experimental conditions.Ripple oscillations, paired with immunohistochemical (IHC) methods (described below), were used to identify electrode placement. For ripple measurements, periods in which subjects were immobile were isolated and the LFPs from these periods were filtered in the ripple range (100–300 Hz). The channel with the maximum amplitude of putative ripple events was identified as the pyramidal cell layer G.To aid in electrode localization, at the end of all experiments for each animal, a small amount of current was passed through the channel corresponding to the bottom-most electrode pad of each electrode (5 μA for 5 s) to create a small lesion at the most ventral recording location of that probe, visible on subsequent IHC processing. Three hours after creating the lesion, animals were euthanized with Fatal Plus  and underwent transcardial perfusion with 4% paraformaldehyde. Heads with electrodes remaining implanted were submerged in 4% paraformaldehyde for 72 h. Brains were then carefully removed, rinsed in PBS, and sectioned at 40 μm on a vibratome.For IHC, brain sections were rinsed in PBS, boiled in deionized water for 3 min, and blocked for at least 1 h in 3–5% normal goat serum/0.01% Tween 20 PBS. Sections were then incubated in mouse anti-RGS14  and rabbit anti-WFS1  primary antibodies. Sections in which an electrode track was visible were incubated in rabbit anti-IBA1 , a marker for activated microglia H. AntiboAmigo2-icreERT2 (10 Amigo2-icreERT2+ and nine Amigo2-icreERT2–) mice were used. Amigo2-icreERT2 mice were arbitrarily selected from individual litters. No specific method of randomization was used to assign groups. Whenever possible, Cre+ and Cre– mice from the same litter were used. Animals were housed with same-sex littermates during weaning and individually housed for the duration of experiments following virus infusion. Pre-established criteria for excluding mice from analysis included (1) missed electrode implantation, (2) equipment failures during testing, and (3) insufficient investigation of experimental stimuli, with a minimum of 10 s of investigation per recorded session required to be included in the analysis.For all experiments presented, 19 male N when indicating the number of animals that were used for the experiment. Statistical tests used for each experiment are presented in the text. Statistical significance was based on p = 0.05. All error bars in graphs represent SEM. For statistical analysis of γ oscillatory powers presented in t tests to explore the source of the observed interaction effect. Statistical analysis of theta powers was performed separately from γ oscillatory powers due to the larger values and greater variability in theta power we observed during non-mobile investigative behaviors. Theta powers for each layer were compared between vehicle and CNO conditions using paired t tests. Locomotor velocity during LFP sampling and follow-up analyses of γ powers on presentation of each stimulus separately . This mouse line enabled us to selectively target and inhibit activity of CA2 pyramidal neurons in vivo and measure the resulting network and behavioral effects.To gain selective genetic access to molecularly-defined CA2, we used a previously-generated tamoxifen-inducible, Cre-dependent mouse line, icreERT2 . InfusioicreERT2 A–E with th RGS14 A,B and lth RGS14 C,D. mCheAmigo2-icreERT2+ mice has been shown to reduce slow γ power in the pyramidal cell layer of CA1 during running , frequency , stimulus type , and drug condition , and results are presented in Inhibition of CA2 via expression and activation of hM4Di in CA2 pyramidal cells of  running . Therefoe probes F. For eaAmigo2-icreERT2+ mice, we found significant main effects of layer  = 25.31, p = 0.000; N = 8) and frequency  = 214.33, p = 0.000; N = 8) as well as an interaction effect between layer and frequency  = 45.20, p = 0.000; N = 8). Additionally, we found a significant interaction among layer, frequency, and drug  = 3.58, p = 0.034; N = 8). By contrast, the RM-GLM revealed no main effect of stimulus  = 0.02, p = 0.887; N = 8) nor any significant interactions involving stimulus. Nevertheless, given our a priori hypothesis that inhibition of CA2 may alter γ oscillatory power in a stimulus-specific manner, we followed up by separating the data according to stimulus and compared γ power on either vehicle or CNO treatment for each stimulus exposure (In t(7) = 2.55, p = 0.038; N = 8; t(7) = 3.09, p = 0.018; N = 8; t(6) = 1.19, p = 0.278; N = 7; t(6) = 0.34, p = 0.745; N = 7; t(7) = 4.55, p = 0.003; N = 8; t(7) = 7.36, p = 0.0002; N = 8; t(6) = 3.35, p = 0.015; N = 7; t(6) = 1.90, p = 0.107; N = 7; Because we found no main or interaction effects of stimulus type on γ oscillatory power in our experiments, we first collapsed data collected on investigation of social and object stimuli and asked how CNO versus vehicle treatment affected slow and fast γ power in each layer of CA1 , 4. For Amigo2-icreERT2+ for recordings obtained from SO (t(7) = 1.45, p = 0.192; t(7) = 0.466, p = 0.655; t(6) = 0.999, p = 0.356; t(6) = 1.52, p = 0.180; We also measured theta powers and found no significant difference in theta power between CNO or vehicle treatment in Amigo2-icreERT2– mice, we found a significant main effect of layer  = 24.04, p = 0.000; N = 7) and frequency  = 57.98, p = 0.001; N = 7), as well as an interaction effect between layer and frequency  = 25.11, p = 0.001; N = 7). We found no main effect of stimulus type or drug and no interactions involving stimulus type or drug  = 0.74, p = 0.493; N = 6; t(6) = 0.33, p = 0.756; N = 7; t(6) = 0.49, p = 0.643; N = 7; t(6) = 1.26, p = 0.253; N = 7; t(5) = 0.91, p = 0.405; N = 6; t(6) = 0.96, p = 0.373; N = 7; t(6) = 0.59, p = 0.579; N = 7; t(6) = 67, p = 0.527; N = 7; For Amigo2-icreERT2– mice for recordings obtained from SO (t(5) = 1.16, p = 0.299; t(6) = 0.351, p = 0.737; t(6) = 0.438, p = 0.677; t(6) = 0.226, p = 0.829; Theta power was also not significantly different between CNO or vehicle treatment in Amigo2-icreERT2+ animals  = 0.000629, p = 0.981; main effect of treatment: F = 1.083, p = 0.333) or Amigo2-icreERT2– animals  = 0.1282, p = 0.733; main effect of treatment: F = 0.02,376, p = 0.883).We also compared mean velocity of the animals during the times used for LFP analysis for each treatment and stimulus presentation and found no significant differences in locomotor velocity for either a priori hypothesis before beginning this study was that inhibition of CA2 may alter γ oscillatory power in a stimulus-specific manner. Therefore, as a follow-up analysis to that presented in Our Amigo2-icreERT2+ animals, we found that slow γ power was significantly reduced in the pyramidal cell layer upon CNO treatment compared with vehicle treatment selectively during novel animal investigation; slow γ power was not significantly reduced following CNO treatment during novel object investigation but was significantly reduced during novel animal investigation  = 9.5, p = 0.0178; main effect of stimulus: F = 2.357; p = 0.1686; interaction: F = 1.099, p = 0.3293; novel animal: p = 0.0136, novel object: p =0.1063). In the SO layer, slow γ power was overall significantly reduced following CNO treatment compared with vehicle treatment but each stimulus alone did not significantly reduce slow γ power  = 6.477, p = 0.0384; main effect of stimulus: F = 0.5069; p = 0.4995; interaction: F = 0.4712, p = 0.5145; novel animal: p = 0.1015, novel object: p = 0.3913). Slow γ power was not significantly reduced by CNO in either SR  = 1.416, p = 0.2790; main effect of stimulus: F = 0.08,567; p = 0.7796; interaction: F = 0.432, p = 0.5354) or SLM  = 0.1542, p = 0.71 081; main effect of stimulus: F = 0.02,751; p = 0.837; interaction: F = 0.2365, p = 0.6440).For Amigo2-icreERT2+ animals, fast γ power in the pyramidal cell layer was also significantly reduced following CNO treatment. Interestingly though, whereas slow γ power reduction was selective for social stimulus exposure, fast γ power in the pyramidal cell layer was significantly reduced during both novel social stimulus and novel object stimulus exposure  = 55.47, p = 0.0001; main effect of stimulus: F = 0.003674; p = 0.9534; interaction: F = 0.9945, p = 0.3519; novel animal: p = 0.0109, novel object: p = 0.0021). In the SO and SR layers, fast γ power was overall significantly reduced following CNO treatment compared with vehicle treatment  = 20.82, p = 0.0026; main effect of stimulus: F = 0.001639, p = 0.9688; interaction: F = 0.0263, p = 0.8758; SR: main effect of treatment: F = 11.2, p = 0.0155; main effect of stimulus: F = 0.1965, p = 0.6731; interaction: F = 0.3173, p = 0.5936) but each stimulus alone did not significantly reduce slow γ power . Fast γ power was not significantly reduced by CNO in SLM  = 3.574, p = 0.1076; main effect of stimulus: F = 1.022, p = 0.3511; interaction: F = 4.355, p = 0.0820).Similar to slow γ power in the pyramidal cell layer, among Amigo2-icreERT2– animals  = 0.5475, p = 0.4926; main effect of stimulus: F = 0.01,439 p = 0.9092; fast γ: main effect of treatment: F = 0.8265, p = 0.4050; main effect of stimulus: F = 2.188, p = 0.1992), the pyramidal cell layer  = 0.1057, p = 0.7562; main effect of stimulus: F = 0.02,277, p = 0.8850; fast γ: main effect of treatment: F = 0.9247, p = 0.3734; main effect of stimulus: F = 1.999, p = 0.2072), SR  = 0.2309, p = 0.6479; main effect of stimulus: F = 0.1713, p = 0.6933; fast γ: main effect of treatment: F = 0.3515, p = 0.5749; main effect of stimulus: F = 1.321, p = 0.2941) or SLM  = 1.616, p = 0.2507; main effect of stimulus: F = 2273, p = 0.6504; fast γ: main effect of treatment: F = 0.4432, p = 0.5303; main effect of stimulus: F = 0.2385, p = 0.6426).Among  animals , we founAmigo2-icreERT2+ mice, CNO did not significantly affect the amount of time subjects spent investigating novel animals  = 1.91, p = 0.098; N = 8; t(7) = 0.02, p = 0.981). Similarly, in control Amigo2-icreERT2– mice, CNO did not cause a significant change in the amount of time subjects spent investigating novel animals  = 0.32, p = 0.761; N = 7; t(6) = 1.25, p = 0.257). Additionally, we assessed whether subjects showed a preference for either the novel animal or novel object stimulus following either vehicle or CNO administration based on time spent investigating the different stimulus types. Amigo2-icreERT2+ mice did not exhibit a preference for either stimulus type following vehicle  = 0.29, p = 0.784; N = 8; t(7) = 0.67, p = 0.521) administration. Similarly, control Amigo2-icreERT2– mice did not show a preference for either stimulus type following vehicle  = 0.02, p = 0.985; N = 7; t(6) = 1.37, p = 0.221) administration. Additionally, we measured and found no difference in the average distance traveled across experimental days by Amigo2-icreERT2+  = 2.141, p = 0.1149; Amigo2-icreERT2– mice  = 1.772, p = 0.1880; Amigo2-icreERT2+  = 2.082, p = 0.1219; Amigo2-icreERT2– mice  = 1.528, p = 0.2394; Based on our findings of reduced slow and fast γ power on acute inhibition of CA2 pyramidal cells during investigation of novel stimuli, we asked whether the effects of CNO observed on neuronal oscillations were accompanied by changes in behavior that may have been responsible for changes in neuronal activity. First, we assessed whether CNO administration alone caused changes in the time subjects spent investigating the different types of stimuli. In Amigo2-icreERT2 mice and examined neuronal oscillations in the theta, slow γ and fast γ frequency ranges while subjects freely explored novel juvenile conspecifics or novel inanimate objects that were confined to a single position in space. We then chemogenetically inhibited CA2 pyramidal cells using hM4Di to determine whether CA2 contributes to the organization of theta, slow γ or fast γ oscillations in CA1 during investigation of novel stimuli. We found that acute inhibition of CA2 pyramidal cells reduced γ power in a layer- and frequency-dependent manner, with effects limited to the dendritic domains targeted by CA2 axons. In addition, analysis of changes in γ power by each type of stimulus revealed a stimulus-specific effect of silencing CA2 on γ oscillatory power; CA2 inhibition decreased slow γ power in the pyramidal cell layer during investigation of novel animals but not novel objects. Interestingly, CA2 inhibition decreased fast γ power in the pyramidal cell layer during both novel animal and novel object investigation. No significant change in γ power was detected in SLM, the only layer not targeted by CA2 neurons. In addition, we observed no significant change in theta oscillatory power on CA2 inhibition. These findings reveal three novel points about the role of CA2 in organizing hippocampal oscillatory networks.In this study, we recorded LFPs from across CA1 layers in adult, male First, CA2 modulates γ activity in the SR and SO dendritic layers of CA1 in addition to the pyramidal cell layer but does not affect SLM. The generation of independent, layer-specific γ oscillations in CA1 has previously been shown to depend on selective axonal targeting from upstream regions that modulate firing of CA1 GABAergic interneurons . CA2 pyrSecond, these experiments suggest that in addition to regulating slow γ oscillations, CA2 neuronal activity may serve to amplify fast γ oscillations in CA1. Slow γ oscillations in CA1 have previously been shown to rely on CA3 Schaffer collateral input . In addiThird, we provide evidence that slow γ oscillations are decreased in response to CA2 silencing during investigation of novel social stimuli but not novel object stimuli. CA2 has been shown to be required for social recognition memory , but wheGiven the previous finding that chemogenetic modulation of CA2 neuronal activity affects slow γ power in the CA1 pyramidal cell layer , we favoBased on our physiological findings, we asked whether the observed reductions in γ power were accompanied by changes in behavior that may have produced the changes in neuronal oscillations. We measured time spent investigating the different types of stimuli following treatment with CNO or vehicle and found no difference in the amount of time mice spent investigating either stimulus type following either treatment. Because we were interested in the role of CA2 in hippocampal oscillatory networks and the electrophysiological effects of targeted CA2 manipulation, we selected a behavior that did not require behavioral training. For this reason, the lack of a significant effect of CA2 inhibition on stimulus investigation times is not surprising, as we used a passive exposure paradigm in which subjects could freely explore the environment of their own volition, with each session separated by 24 h. In contrast, social recognition paradigms typically measure investigation time of social stimuli on repeated exposures taking place over the course of minutes to hours . These dIn addition, we note that the dose of CNO we used (3 mg/kg) was lower than that used in previous study of CA2 and social memory (10 mg/kg; In conclusion, we found that CA2 neuronal activity contributes to slow and fast γ oscillations in CA1 in a dendritic layer-dependent manner. Slow γ oscillations were preferentially impacted by CA2 silencing during novel animal investigation over novel object investigation, whereas fast γ oscillations were affected by CA2 silencing during either novel animal or novel object investigation. These findings support a role of CA2 neuronal activity in hippocampal γ oscillations and processing of social and contextual information.


We also propose other potential targeting strategies for developing more specific and effective MDMX inhibitors for cancer therapy.The oncogene MDMX, also known as MDM4 is a critical negative regulator of the tumor suppressor p53 and has been implicated in the initiation and progression of human cancers. Increasing evidence indicates that MDMX is often amplified and highly expressed in human cancers, promotes cancer cell growth, and inhibits apoptosis by dampening p53-mediated transcription of its target genes. Inhibiting MDMX-p53 interaction has been found to be effective for restoring the tumor suppressor activity of p53. Therefore, MDMX is becoming one of the most promising molecular targets for developing anticancer therapeutics. In the present review, we mainly focus on the current MDMX-targeting strategies and known MDMX inhibitors, as well as their mechanisms of action and  MDM2 hain vivo –12, as win vivo , 14, MDMin vivo –18.The p53-MDM2 pathway has been extensively investigated during the past three decades and has been found to play critical roles in cancer initiation and progression –21. BecaThe MDMX protein consists of 490 amino acids with four main conserved domains, including the N-terminal p53-binding domain, the central acidic domain, the zinc-finger domain, and the C-terminal RING domain  15, 22), 2215, 2in vivo , but not its oncogenic MDMX-S variant is significantly associated with the responses of ovarian cancer patients to chemotherapy  is increased and correlated with high tumor grade and poor overall survival   of CLL patients (MDMX amplification has been reported in both soft tissue sarcomas (17%) and osteosarcomas (35%) –77. Bartsurvival . Lenos eotherapy . Further disease . The oveia (CLL) , acute mia (CLL) , mantle ia (CLL) , and pedia (CLL) . Liu et mutation . Importapatients .MDMX-S overexpression and a highly significant downregulation of MDMX mRNA in papillary thyroid carcinomas, which has been correlated with the multifocality of tumors and advanced tumor stage  and has been correlated with the poor outcomes of patients with this disease . Ach et or stage . In addior stage , head anor stage , and retor stage . Several cancers –91. Howein vitro and in vivo activities and mechanisms of action.There is a lot of evidence indicating that cancer often upregulates the expression of MDMX to dampen the tumor-suppressor function of p53. Inhibiting MDMX helps restore the tumor suppressor activity of p53 as well as repress the oncogenic function of MDM2. Therefore, MDMX has recently been demonstrated as a promising therapeutic target for cancer therapy . Severalin vitro and in vivo (KD = 2.3 nM)  . SAH-p53 mSF-SAH . AlthougKi = 5.5 μM) using the FP-based assay. However, the anticancer efficacy of compound B1 has yet to be determined  by using a fluorescence polarization (FP)-based MDMX-p53 binding assay as well as a cell-based assay and identified 11 representative compounds . Among ttermined .KD = 450 nM), prevents the formation of p53-MDMX complex, and activates p53 without causing DNA damage  xenograft tumors in vivo  and identified a lead compound CTX1, which induces strong p53-dependent activation in MCF-7 cells expressing a high level of MDMX . FurtherA damage . CTX1 al in vivo . In addi in vivo . Furtherin vitro and in vivo. Optimization of p53 stapled peptides has been considered as a reasonable strategy to discover effective p53 activators, which leads to the identification of ATSP-7041 and ALRN-6924  and effectively disrupts the p53-MDM2/MDMX bindings, thereby activating p53 signaling in p53 wild-type cancer cells. ATSP-7041 has been found to induce cell cycle arrest in G1 and G2/M phase and apoptosis in vitro and suppress xenograft tumor growth in vivo in a p53-dependent manner . It has further been found that ALRN-6924 inhibits the p53-MDM2/MDMX interactions and activates p53, thus inhibiting cell proliferation and clonogenic capacity and causing cell cycle arrest and apoptosis in AML cells harboring wild-type p53 , which decreases both the MDMX mRNA and protein levels through repressing activity . Furtheractivity . It has in vitro . Pishas in vitro . Howeverin vitro . In addiin vitro . Furtherf XI-011 . Furtherin vitro and suppresses tumor growth in an H460 xenograft mouse model in vivo. The molecular mechanism studies have shown that luteolin up-regulates the expression of miR-34a-5p in NSCLC cells, which consequently leads to the down-regulation of its downstream target MDMX as well as the upregulation of p53 and p21   . Furthertosis 3) .in vitro and in vivo, with minimal host toxicity  have been used to efficiently modulate splicing . Dewaele in vivo .MDMX and activates the p53 pathway. Further studies have shown that GSK3203591 inhibits cancer cell growth and survival in vitro and suppresses tumor growth in lymphoma xenograft mouse models in vivo (Protein arginine methyltransferase 5 (PRMT5) has recently been identified as a master regulator of splicing, which regulates MDMX abundance by controlling its alternative splicing . Herein, in vivo .Given that the formation of MDM2-MDMX complex stabilizes MDM2 protein, enhances its E3 ubiquitin ligase activity, and promotes the ubiquitination and degradation of MDMX, the MDM2-MDMX binding has been considered as a promising target for cancer therapy. Ling et al. have reported that FL118, a previously discovered inhibitor of survivin expression inhibits p53 ubiquitination but promotes MDMX protein degradation . They hain vitro and in vivo  induces cancer cell apoptosis and synergistically enhances nutlin-3-induced apoptosis in a p53-dependent manner . 17AAG hin vitro and in vivo. Their main focus has been on the design and development of p53-MDMX binding inhibitors by targeting the p53-binding pocket of MDMX, such as SAH-p53-8  , XI-011 MDMX-S mRNA is often observed, the endogenous MDMX-S protein is undetectable in any normal or cancer cell lines, which implies that the endogenous MDMX-S may be a very unstable protein and the switch from MDMX-FL to MDMX-S may play a critical role in controlling the protein level of MDMX-FL and activating p53 in normal and/or cancer cells  binding, and p53-HIPK2 (Homeodomain-interacting protein kinase 2) functional interaction, and promotes p53-mediated apoptosis  and its mitochondrial localization, facilitates the p53Ser46poptosis , 133. Bepoptosis . It has poptosis , 136. AlIn conclusion, the accumulating knowledge about the roles of MDMX in the p53-MDM2/MDMX loop, its p53-independent oncogenic functions, and the regulatory mechanisms for MDMX expression and protein stability may provide new opportunities to develop novel MDMX inhibitors. A better understanding of these MDMX inhibitors, especially their selectivity, efficacy, pharmacological, and toxicological properties is critical for moving them forward from bench to clinical trials.J-JQ conceptualized the manuscript. D-HY, Z-YX, SM, LY, X-DC, and J-JQ collected the literature, wrote the manuscript, and made the figures. J-JQ edited and made significant revisions to the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Matrix metalloproteinase‐9 (MMP‐9), or gelatinase B, has been hypothesized to be involved in the progression of atherosclerosis. In the arterial wall, accumulated macrophages secrete considerable amounts of MMP‐9 but its pathophysiological functions in atherosclerosis have not been fully elucidated. To examine the hypothesis that macrophage‐derived MMP‐9 may affect atherosclerosis, we created MMP‐9 transgenic (Tg) rabbits to overexpress the rabbit MMP‐9 gene under the control of the scavenger receptor A enhancer/promoter and examined their susceptibility to cholesterol diet‐induced atherosclerosis. Tg rabbits along with non‐Tg rabbits were fed a cholesterol diet for 16 and 28 weeks, and their aortic and coronary atherosclerosis was compared. Gross aortic lesion areas were significantly increased in female Tg rabbits at 28 weeks; however, pathological examination revealed that all the lesions of Tg rabbits fed a cholesterol diet for either 16 or 28 weeks were characterized by increased monocyte/macrophage accumulation and prominent lipid core formation compared with those of non‐Tg rabbits. Macrophages isolated from Tg rabbits exhibited higher infiltrative activity towards a chemoattractant, MCP‐1 in vitro and augmented capability of hydrolysing extracellular matrix in granulomatous tissue. Surprisingly, the lesions of Tg rabbits showed more advanced lesions with remarkable calcification in both aortas and coronary arteries. In conclusion, macrophage‐derived MMP‐9 facilitates the infiltration of monocyte/macrophages into the lesions thereby enhancing the progression of atherosclerosis. Increased accumulation of lesional macrophages may promote vascular calcification. The blot was rehybridized with a 32P‐labelled human β‐actin probe (Clontech Laboratories) to confirm that equal amounts of RNA were loaded in each lane. To evaluate MMP‐9 protein expression and enzymatic activity, we collected elicited peritoneal macrophages from the peritoneal cavity 4 days after injection of 4% Brewer's thioglycollate broth, as described previously.6) from either Tg or non‐Tg rabbits (n = 5 for each group) were incubated in serum‐free 1640 medium with or without phorbol 12‐myristate 13‐acetate (Sigma‐Aldrich Com) (50 ng/mL) for 48 hours, and the conditioned media were then collected for Western blotting and gelatin zymographic analysis.2, 1 μmol/L ZnCl2, 0.2 mmol/L NaN3 and 0.05% BRIJ‐35) at 37°C for 36 hours. After that, the gels were stained by Coomassie Brilliant Blue (Wako) till the bands were visualized. Areas of enzymatic activity showed as clear bands over the mazarine background. Gels were scanned, and the density of digested bands was quantified with ImageJ software . To measure the enzymatic activity of gelatinases in the aortic tissue directly, we examined gelatin activity of aortic arch homogenate (n = 4 for each group) with an EnzChek Gelatinase kit . 100 μg of crude protein of each sample was incubated with quenched fluorescein‐conjugated gelatin at 37°C in darkness. Active enzymes cleave the fluorescent‐labelled gelatin and the rate of proteolysis was expressed by fluorescence intensity. To verify the gelatinase activity specificity, aortic arch homogenate was incubated with MMP‐2/9 inhibitor (10 μmol/L)  for 1 hour at 37°C before mixed with quenched fluorescein‐conjugated gelatin. Fluorescence was measured at multiple time‐points during reaction using a SpectraMax Microplate Reader (Molecular Devices) (absorption 490 nm/emission 515 nm). We also performed in situ gelatinolytic activity using frozen sections of aortic arch as previously reported. 8‐µm‐thick cryosections were air‐dried for 10 minutes. After that, sections were washed with PBS to remove traces of OCT. Quenched fluorescein‐conjugated gelatin (Molecular Probes) was used as the substrate. The gelatin (20 µg/mL), which was dissolved in reaction buffer with 10% low gelling agarose (Sigma‐Aldrich), was dropped on the top of sections and incubated with reaction buffer in a dark humidity chamber at 37°C for 6 hours. 5 mmol/L EDTA in reaction buffer was used to block gelatinase activity. The sections were then rinsed with PBS and stained with DAPI for staining nuclei. Slides were then mounted with a cover glass using an aqueous agent.To examine the tissue expression of MMP‐9 in Tg and non‐Tg rabbits, Northern blotting was performed as described previously.2.3Tg rabbits along with sex‐ and age‐matched non‐Tg littermates were used for the following experiment. In the first experiment, rabbits  were fed a diet containing 0.5% cholesterol and 3% soybean oil for 16 weeks. The lesions of atherosclerosis formed in these rabbits were mainly those of so‐called early‐stage lesions (fatty streaks), and thus, it was amenable to evaluate the effects of increased MMP‐9 expression on the initiation of atherosclerosis. In the second experiment, rabbits  were fed the same cholesterol‐rich diet for 28 weeks. Long‐duration hypercholesterolaemia in the second experiment in comparison with the first experiment was expected to induce more advanced atherosclerotic lesions, such as fibrous plaques and complicated plaques, in these rabbits.2.4en face stained by Sudan IV solution for quantitative analysis of the gross atherosclerotic lesion area as described previously.At the end of cholesterol diet feeding, all rabbits were euthanized by injection of sodium pentobarbital solution (100 mg/kg BW) through an ear vein. The aortas were 2.5http://www.sciencedirect.com/science/article/pii/S0163725814001855). The blocks were cut into 4 serial sections (3 μm thick) at 50 μm intervals, and in total, 4 cuts were conducted. For 4 sections collected from each cut were stained for HE, EVG, and von Kossa or immunohistochemically stained with mAb (RAM11) against macrophages. The lesion size was quantified by EVG‐stained specimens and expressed as stenosis % (lesion area/ coronary lumen area). Calcification and Mϕ areas were quantified as aortic lesion analysis described above and expressed as mm2.To assess coronary atherosclerosis, all hearts of both male and female rabbits of the second experiment were cut into five blocks and the block‐I, which contains the main trunk of the left coronary artery, was used for coronary lesion analysis. The whole protocol for heart sections was described in details elsewhere2.6To evaluate the presence of cellular apoptosis in the lesions, serial sections of paraffin‐embedded aortas were either immunohistochemically stained by Abs against macrophage, caspase‐3 or by TUNEL (TdT‐mediated dUTP nick end labelling) method using ApopTag peroxidase in situ apoptosis detection kits . For TUNEL staining, all the procedures followed the manufacturer's instruction. Briefly, sections of the aortic arch were deparaffinized and rehydrated through xylene and graded ethanol to distilled water. Then, specimens were treated in 3.0% hydrogen peroxide in PBS solution for 5 minutes at room temperature for quenching endogenous peroxidase. Terminal deoxynucleotidyl transferase (TdT) with digoxigenin labelled deoxyuridine triphosphate was added and incubated in a humidified chamber at 37°C for 30 minutes and then washed with PBS buffer. After that, specimens were incubated with peroxidase‐conjugated anti‐digoxigenin Ab in a humidified chamber for 30 minutes at room temperature and then washed 4 times in PBS. After that, AEC (Nichirei Bioscience) was added for visualizing the nucleotides, and nuclei were stained with haematoxylin. The negative control was conducted by using PBS to replace TdT Figure .2.7Sections of aorta arch were deparaffinized and rehydrated through graded ethanol to distilled water. The slides were then incubated with TRAP staining solution which contained 0.1 mg/mL naphthol AS‐MX (Sigma‐Aldrich) and 0.08 mg/mL fast red violet LB salt (Sigma‐Aldrich) in the presence of 8 mmol/L sodium tartrate and 30 mmol/L sodium acetate (pH 5.0) at 37°C for 2 hours. After rinsing in distilled water, the slides were mounted with aqueous mounting medium.2.82), the number of macrophages that penetrated the membranes was counted in 10 high‐power fields randomly from each well.To examine the effects of MMP‐9 enzymatic activity on macrophage migration and matrix digestion, we performed chemotaxis analysis and granuloma assays. Chemotaxis assays for alveolar macrophages isolated from Tg and non‐Tg rabbits (n = 4 for each group) were performed using Biocoat cell culture inserts coated with laminin . The lower compartments were loaded with the same medium containing human recombinant monocyte chemoattractant protein‐1 (MCP‐1) (Pepro Tech EC) at 10 ng/mL. After 48 hours of incubation  by the Mann‐Whitney U test. In all cases, significance was set as a P‐value <.05.Statistical analysis was performed using SPSS 16.0 software. All data are expressed as mean ± SEM. Statistical analysis was performed for parametric data by Student's 33.1We generated 2 Tg founder rabbits as shown by Southern blotting analysis Figure B. One fo3.2We first examined whether MMP‐9 overexpression affected the early‐stage lesion formation of atherosclerosis in rabbits fed a cholesterol diet for 16 weeks. As shown in Figure 3.3In the second experiment, we fed rabbits with a cholesterol diet for 28 weeks to examine whether increased MMP‐9 expression influenced the advanced lesion formation. Compared with the experiment 1 in which the lesions were smaller in size and mainly consisted of the fatty streaks, the lesions of the experiment 2 were more extensive and predominately by advanced lesions, such as fibrous plaques and atheroma (types IV‐V lesions).One of the striking features of advanced lesions observed in Tg rabbits was the prominent vascular calcification Figure . AlthougIn addition to the severe advanced lesions associated with prominent calcification in Tg rabbits described above, macrophages frequently expanded the deep parts of the tunica media or even the adventitial layers Figure .3.4P < .01) than those of non‐Tg rabbits   and MMP‐12 (elastase) because Tg rabbits expressing either MMP‐1. Accumulation of macrophages in the lesions even extended to the deeper medial tunica or even the adventitia. Although it is still unknown whether these adventitial macrophages come from the inside or outside, it is most likely that they develop as a result of ‘outside in’ rather than ‘inside out’ migration. Regardless of this assumption, emigration of monocytes/macrophages requires the degradation of the ECM and increased MMP‐9 activity in Tg macrophages undoubtedly leads to enhancement of ECM degradation thereby facilitating the influx of monocyte/macrophages into the lesions.In the second experiment, rabbits were fed a cholesterol diet for 28 weeks and developed greater and more advanced lesions (than experiment one) which enabled us to examine whether increased MMP‐9 affects the progression of atherosclerosis. Although the aortic arch lesions stained by Sudan IV became almost saturated at 28 weeks, gross lesion areas were significantly increased in female  Tg rabbits. The molecular mechanism for this gender effect of MMP‐9 is still unknown but it will be interesting to investigate whether sex hormones interact with MMP‐9 in future. Nevertheless, lesional macrophages were significantly increased in both aortic and coronary atherosclerotic lesions in both male and female Tg rabbits compared with non‐Tg rabbitsIn spite of this, one of the most striking findings observed in the current study was the demonstration of remarkable calcification in the aortic and coronary lesions in Tg rabbits, which mimicked human advanced atherosclerotic lesions with calcification.In conclusion, we have successfully created Tg rabbits that overexpress MMP‐9 specifically in macrophage lineage. Increased expression of MMP‐9 in macrophages not only enhances the formation of advanced atherosclerotic lesions in Tg rabbits, but also leads to a marked vascular calcification. Therefore, macrophage‐derived MMP‐9 in the arterial wall may exert other physiological functions beyond its classical gelatinase activity. Although the molecular mechanisms remain yet to be clarified, it will be interesting to investigate in the future whether inhibition of MMP‐9 functions as a therapeutic method to inhibit vascular calcification.None.JF and Y.EC designed the study; KN, SK and FM created and bred Tg rabbits. YC, ABW, BN, LC, HS, TK, YY, JZ, JL and EL performed experiments, collected, analysed and interpreted the data. YC and JF wrote the paper. All authors had final approval of manuscript submission. Click here for additional data file. Click here for additional data file.


Calcium imaging is the most common approach to studying dynamic changes in the cytoplasmic Ca2+ content. Here, we used mature ‘Fuji’ apples  to obtain viable protoplasts from flesh tissue cells by enzymatic hydrolysis; then, three small-molecule fluorescent probes  were loaded into the protoplasts. All three Ca2+ fluorescent probes successfully entered the cytoplasm but did not enter the vacuole. Both the Ca2+ chelator (EGTA) and Ca2+ channel blocker (La3+) reduced the fluorescence intensity in the cytoplasm. The calcium ionophore A23187 increased the fluorescence intensity in the cytoplasm at 5 µmol/L but decreased it at 50 µmol/L. Additionally, A23187 reversed the fluorescence intensity in the cytoplasm, which was decreased by La3+. Ionomycin is also a calcium ionophore that can increase the fluorescence intensity of calcium in the cytoplasm. These results suggest that small-molecule Ca2+ fluorescent probes can be used to detect changes in cytosolic calcium levels in the cells of fruit flesh tissue. In addition, the optimum concentration of fluo-8/AM was determined to be 5 µmol/L. This was the first time that protoplasts have been isolated from apple flesh tissue cells and small-molecule fluorescent probes have been used to detect calcium in the cytoplasm of flesh tissue cells. This study provides a new method to study calcium signal transduction in fruit flesh tissue.Cytosolic Ca Thus, a low calcium content reduces fruit firmness and shortens the length of the storage period. A disorder in cell calcium metabolism can cause apple bitter pit5, brown spot disease6, and cock spot in Pyrus serotina7. In vegetable crops, tomato umbilical rot is also caused by a calcium metabolism disorder8. These physiological disorders severely degrade fruit quality and cause serious economic losses to growers9. Therefore, growers often supplement fruit with calcium. Spraying calcium early during fruit growth and soaking in calcium at postharvest can improve fruit firmness10, reduce the incidence of bitter pit11 and cock spot7, delay fruit senescence and softening, and extend the storage period2.Calcium influences many fruit qualities3. Spraying calcium on the fruit skin surface is not an effective way to prevent litchi fruit cracking12, nor can it reduce the incidence of apple bitter pit, as was shown in a study in which calcium spraying was repeated five times over the entire apple fruit growth period13. Moreover, other studies have shown that the calcium content of bitter-pit fruit is not lower than that of healthy fruit14. The main reason for these conflicting results is that the role of calcium in flesh tissue cells is not clear15. In the cytoplasm, Ca2+ acts as a signaling ion that mediates a variety of cell growth and development processes16.However, calcium supplementation is not always effective. Studies have shown that calcium spraying or soaking does not always increase fruit firmness2+ in living cells with good spatial and temporal resolution18. At present, there are two methods for intracellular calcium imaging in living cells: one uses small-molecule fluorescent probes, and the other uses FRET (Förster Resonance Energy Transfer)—based genetically encoded sensors (GECIs)19. Although GECIs have many advantages, a stable transgenic system for fruit trees is difficult to establish, and the growth period of fruit trees is inconveniently long;20 furthermore, the weak fluorescence of single cells is not conducive to overcoming background noise, and the method is susceptible to interference from endogenous calmodulin and other shortcomings21.Calcium imaging has been demonstrated to be a powerful method for observing the dynamic changes in intracellular Ca2+-specific selectivity and are noninvasively loaded by esterification incubation, which is flexible, rapid, and not cytotoxic22. Fluo-4/AM was successfully loaded into the pollen tube of Pyrus pyrifolia20 and petunia23 as well as into the guard cells24 and root hairs26 of Arabidopsis. Fluo-8/AM can also be used to detect dynamic calcium in guard cells27.Small-molecule fluorescent indicators, such as fluo-4/AM and fluo-8/AM, show Ca28. Moreover, apples are economically important worldwide and a healthy food29. Today, ‘Fuji’ apples are one of the most popular sweet apple varieties in the world and are commercially grown in Japan, China, the United States, and Australia. Especially in China, ‘Fuji’ is the main planting variety, and its yield and cultivated area account for more than 60% of the total apple production and total cultivated area30. Here, we first obtained viable protoplasts from ‘Fuji’ apple flesh tissue cells and then detected Ca2+ in the cytoplasm with a small-molecule calcium fluorescent reagent.At present, the study of plant calcium imaging mainly focuses on pollen tubes, root hairs, and guard cells. There are few reports on calcium fluorescence staining of flesh cells. Calcium may be the most important mineral determining the quality of fruits, especially apples and pears, because they are commonly stored for long periods of time2+ that can also be combined with a Ca2+ fluorescence indicator;31 thus, it was difficult to determine whether fluorescence was from the cell wall or from the cytoplasm.Apple fruit flesh was manually cut as thin as possible with a surgical knife. The slices did not display fluorescence prior to loading with fluo-8/AM Fig. , which iWe used an enzymatic method to obtain protoplasts from individual apple flesh cells Fig. . We meas2+. Protoplasts showed no fluorescence when no Ca2+ fluorescent indicator was loaded into them  to measure cytoplasmic Cahem Fig. . On the hem Fig. . Furtherhem Fig. . Howeverhem Fig. , as rhodP < 0.001)   Fig. . However01) Fig. , but the05) Fig. . We calc05) Fig. , mainly 05) Fig. . Althoug2+ chelator (EGTA)34 was added to the protoplasts with fluo-8/AM at final concentrations of 1 mmol/L or 10 mmol/L. EGTA is a chelating agent for Ca2+ that can significantly reduce fluorescence intensity  showed a slight decrease in fluorescence intensity within 25 min 42 loaded fluo-3/AM at a low temperature (4 °C) so that it would enter the root tip cells. Similarly, Qu et al. (2012)43 used fluo-3/AM, but at a high temperature (37 °C), to image the inside of pollen tubes. These methods aimed to reduce the activity of esterases on the cell membrane. To overcome the barrier of the cell membrane, the microinjection method has been used to directly inject the fluorescent probes into the cell. The cell wall is a pool of Ca2+ that interferes with the fluorescent intensity of Ca2+ in the cytoplasm when loaded with a fluorescent probe31. To avoid this interference, the microinjection method is also used to avoid the cell wall44. However, this method is very complicated and slow33. In a previous study, we used cell lysates to slightly degrade the cell membrane without reducing cell viability and allowed the calcium fluorescent probe to enter the pollen tube20. In the present study, we successfully removed the cell wall of flesh cells by enzymatic hydrolysis and loaded fluorescent probes into protoplasts at a high temperature (37 °C). This method did not affect the viability of the protoplasts. Additionally, we loaded fluo-8/AM into the protoplasts at low temperature (4 °C) but failed to stain cytoplasmic Ca2+  ester, and the dye should be made neutral so that it can cross the cell membrane33. In other words, it is easy for the probe to accumulate in the vacuole. In the cytoplasm, calcium concentrations are only 100–200 nmol/L, which is much lower than the concentration in the vacuole45, where it ranges from 0.1 to 10 mmol/L45. Nonetheless, the vacuole showed almost no fluorescence in this study, a finding that suggests that none of the three calcium fluorescent probes used were compartmentalized in the protoplasts. In addition, calcium fluorescence was uniformly distributed in protoplasts without vacuoles  but decreased it at high concentrations (50 µmol/L). As a calcium carrier52, it has been reported that ionomycin can increase the concentration of calcium in the cytoplasm53, and in this study, ionomycin increased the fluorescence density of calcium in the cytoplasm of apple pulp cells.Babcock et al.  studied 4. Softening of apple fruits is generally considered an undesirable ripening process because firmer apples tend to have more juice and are crisper crunchier and less mealy than softer apples54. Ca2+ plays a key role in fruit firmness14. Therefore, calcium is added to the fruit before or after harvesting to increase the firmness of the fruit or reduce the speed of fruit softening55. We supplemented Ca2+ and EGTA through the fruit stalk 15 days before the harvest of ‘Golden Del. Reinders’ apple. Exogenous Ca2+ could increase the firmness of the fruit, while EGTA reduced the firmness of the fruit . Exogenous Ca2+ reduces postharvest physiological deterioration by increasing endogenous levels of Ca2+ and inducing the expression of genes related to melatonin biosynthesis after harvest. These effects are reversed by the exogenous application of EGTA56. Our findings support this result. However, Deell et al.  for 30 min. The fluo-8/AM and fluo-4/AM fluorescence intensity was uniformly distributed in the cytoplasm of protoplasts and can be used at 5 μmol/L  to determine the calcium concentration in the cytoplasm. In turn, rhod-2 was granulated in the cytoplasm and can be used to study calcium in the mitochondria. This method can provide technical support for calcium research in fruit and vegetable flesh tissue cells.Malus domestica Borkh. CV. Fuji) apples. The flesh tissue at 1–2 cm under the exocarp (skin) was cut with a scalpel. The flesh tissue was precooled in an embedding solution57, and 80 µm thick slices were cut with a microtome cryostat .We selected disease-free and mature ‘Fuji’ 59 solution containing 0.1% of macerozyme R-10 (w/v) at 28 °C and centrifuged at 70 rpm for 30 min in the dark. The enzyme solution was washed three times with CPW, stirred for 1 h with a magnetic stirrer, and centrifuged at 800 rpm for 3 min; the pulp with single cells was collected.In accordance with our previously published method2·2H2O, 5 mmol/L MES, 0.6 mol/L D-sorbitol, and Tris buffer. The solution was adjusted to pH 5.8. The enzymatic solution was prepared from the following basic solution: 0.004 mg/ml macerozyme R-10 , 0.0001 mg/ml pectolase Y-23 , and 0.002% mg/ml cellulase R-10 . The flesh tissue under the exocarp was cut into small pieces that were 10 × 5 × 1 mm3 in size  was dissolved in DMSO (Dimethyl Sulfoxide) to produce a 1 mg/ml stock solution. One microliter of stock solution was added to 99 μL of DMSO to prepare a working solution. Then, 99 μL of protoplast suspension was placed into a 0.2 ml centrifuge tube, and 1 μL of the FDA working solution was added. The staining was carried out for 5 min at 25 °C in the dark. Before observation, the stained sediments of protoplast suspensions were washed three times with basic solution by centrifugation at 1000 rpm. Then, the viability of the protoplasts was tested under a fluorescence microscope . We selected the light cube of GFP because the excitation wavelength of FDA is 490 nm43. The concentration of the loading solution for preparing different kinds of fluorescent indicators was 0.5 mmol/L; a volume of 99 μL of sliced flesh tissue, single cell or protoplast suspension was placed into 0.5 ml centrifuge tubes, and 1 μL of a loading solution of fluo-4/AM, fluo-8/AM or rhod-2/AM  was added to make the final concentration of the fluorescent dye 5 µmol/L. We loaded the fluorescent dye into the cells for 30 min at 37 °C in the dark. After loading, the dye was washed three times with a basic solution to remove excess fluorescent dye and observed with a fluorescence microscope. Since the excitation wavelength of fluo-4/AM and fluo-8/AM is 490 nm, we selected GFP as the light cube. In turn, RFP was used as the light cube when loading rhod-2/AM because the excitation wavelength of rhod-2/AM is 551 nm.Fluorescent loading solutions were prepared based on our previously published methods3+ reagents so that their final concentrations were 10 mmol/L, 5 μmol/L, 1 μmol/L or 100 μmol/L, respectively. We then continued to observe the changes in calcium fluorescence for 25 min and took photographs every 5 min.We took 18 μL of protoplasts after loading with fluo-8/AM, dropped it onto an adhesive on a concave microscope slide, covered the slide with a coverslip, and observed the calcium fluorescence change in the cells with a fluorescence microscope for 5 min. We then added 2 μL of EGTA (Ethylene Glycol-bis (beta-aminoethyl ether) -N,N,N′,N′-Tetraacetic Acid), A23187, ionomycin, or La20. For final processing, we used Adobe Photoshop CS5 .The fluorescence results were analyzed using Image-Pro Plus 6.0 software  according to our published methodst-test was used to analyze the differences among the experimental groups.Statistical analysis was performed using GraphPad Prism 7.0 software . Data are expressed as the means ± SD. Student’s Supplementary figure S1Supplementary figure S2Supplementary figure S3Supplementary figure S4Supplementary figure S5Supplementary figure S6Supplementary figure S7Supplementary figure S8Supplementary information


Such activity requires the concurrent secretion of large amounts of Ca2+-carrying/transporting proteins acting as a micro-conveyor belt system inside the RER–Golgi. Thus, (fitness increasing) protein secretion is subordinate to Ca2+ removal. Milk with its high content of protein and Ca2+ (60–90 mM vs. 100 nM in unstimulated mammary gland cells) is an extreme example. The sarco(endo)plasmatic reticulum Ca2+-ATPases (SERCAs) and SPCA1a Ca2+/Mn2+ transport ATPases are major players in Ca2+ removal through the Golgi. Both are blocked by the sesquiterpenoid thapsigargin. This strengthens the hypothesis (2014) that endogenous farnesol-like sesquiterpenoids (FLSs) may act as the long sought for but still unidentified agonist(s) for Ca2+-pumps in both the ER and Golgi. A second putative function also emerges. The fusion of both the incoming and outgoing transport vesicles, respectively, at the cis- and trans- side of Golgi stacks, with the membrane system requiring high flexibility and fast self-closing of the involved membranes. These properties may—possibly partially—be controlled by endogenous hydrophobic membrane “fluidizers” for which FLSs are prime candidates. A recent reexamination of unexplained classical data suggests that they are likely synthesized by the Golgi itself. This game-changing hypothesis is endorsed by several arguments and data, some of which date from 1964, that the insect corpus allatum (CA), which is the major production site of farnesol-esters, has active Golgi systems. Thus, in addition to secreting FLS, in particular juvenile hormone(s), it also secretes a protein(s) or peptide(s) with thus far unknown function. This paper suggests answers to various open questions in cell physiology and general endocrinology.The extensive literature dealing with the Golgi system emphasizes its role in protein secretion and modification, usually without specifying from which evolutionary ancient cell physiological necessity such secretion originated. Neither does it specify which functional requirements the secreted proteins must meet. From a reinterpretation of some classical and recent data gained mainly, but not exclusively, from (insect) endocrinology, the view emerged that the likely primordial function of the rough endoplasmic reticulum (RER)–Golgi complex in all eukaryotes was not the secretion of any type of protein but the removal of toxic excess Ca It is less known that Ca2+ is the most abundant toxin on earth, and that exactly this toxicity—at increasing cytoplasmic concentrations to above 100 nM—forms the basis for the cited beneficial effects. The huge Ca2+ concentration gradients over the plasma membrane, ranging from an average of about 1–2 mM extracellularly vs. 100 nM in the cytoplasm of unstimulated cells, in combination with the fact that the plasma membrane is not fully impermeable to Ca2+ (mainly due to Ca2+ channels and pumps), result in a constant drive for Ca2+ to enter the cell. 2+ is gradient-wise distributed not only over the plasma membrane, but intracellularly as well. It illustrates the challenges a eukaryotic cell faces due to the fact that it has to maintain a low concentration of free intracellular Ca2+ in an environment in which the extracellular Ca2+ concentration is at least 20,000 times higher.In cell- and organismal animal physiology, Ca2+ enters the cell, this excess has to be removed as quickly as possible. Small amounts of Ca2+ can be removed by plasma membrane ATPases and functionally related enzymes –Golgi system . If even this mechanism fails, the Ca2+ overload will induce cell death/apoptosis (When for whatever reason(s) excess Capoptosis . Selectipoptosis .2+ is likely to meet with initial skepticism. At first sight, the reason for skepticism seems very logical and understandable, but it is nonetheless erroneous. The “milk example” illustrates why. If people are asked why female mammals after having given birth start producing milk, the almost unanimous answer is: “To feed and provide immune protection to their young!” But if one next asks if the female deliberately plans to engage in milk production, hesitation emerges. This hesitation turns into negation upon asking: “Do the mammary gland cells ‘know’ that they have to produce milk because a young/baby is waiting to be fed?” Of course not. But why is galactopoiesis initiated at all, if there is no “goal” at all to do so? The right answer is that females start secreting milk because of hormonally controlled physiological necessity. The “milk case” illustrates that there is no “goal” in evolution  and has to be eliminated as quickly as possible. The suckling young survives being fed with a potentially toxic nutrient because he or she neutralizes the excess incoming Ca2+ by uploading it in deposits of Ca2+ that are no longer toxic, in particular in the developing skeleton and in intracellular Ca2+ stores, such as the lumina/cisternae of the endoplasmic reticulum, the mitochondria, and Golgi. The mammary gland cells do not succumb under the high Ca2+ concentrations because they make use of the conveyor belt of transport proteins produced in the RER and next secreted by the Golgi, packed into vesicles.The “milk case” raises several cell physiological issues. One concerns the high concentration of Ca0–100 mM . This co2+ homeostasis, the statement that Ca2+ is the most abundant toxic pollutant in the aqueous environment of cells but that it nonetheless became the best communicator may sound strange at first encounter. Calcium is an ambivalent messenger  ] mechanisms and the role of antioxidants   resembleoxidants .2+ homeostasis system with its numerous molecular players  concentration gradient between extracellular and intracellular compartments, which had both signaling function and survival importance. The latter is illustrated by the fact that even relatively (prolonged) moderate increases in cytosolic Ca2+ concentrations above 100 nM are incompatible with life. Thus, the most basic 2+ homeostasis system is to keep cytosolic Ca2+ very lowactivity of the Ca, not higher than 100 nM. Remarkably, which mechanisms control this “keeping Ca2+ low” are only partially understood, in particular, the role of farnesol-like endogenous sesquiterpenoids remains undervalued until to date  are the plasma membrane Ca2+-ATPases (PMCAs) and the sarco/endoplasmic-reticulum Ca2+-transport ATPases (SERCAs with various splice variants). 2+-transport ATPase, PMR1/SPCA Ca2+/Mn2+-transport ATPase  or in cells with a low ATP content such as keratinocytes  changes when the farnesol/juvenile hormone (JH) synthesizing glands, the corpora allata (CA), are inactivated by microsurgical removal. In addition, they also observed this phenotype when the beetles were raised in conditions with a short photoperiod (= less than 12-hour light per day). Other tissues were not investigated, and sites of JH synthesis other than the CA were not known at that time (see later).By electron microscopy, 2+ Homeostasis?” and “From Physiological Necessity”: Which Role For Ca2+ Homeostasis?) interpreted as being physiologically relevant and beneficial for depositing reserves in preparation of hibernation/diapause.The most drastic effect in the fat body cells was on the functioning of the Golgi systems: the secretion of vesicles was totally disturbed. The outgoing transport vesicles no longer fused with the plasma membrane but instead fused with one another, thereby forming large protein bodies  Ca2+ concentration. Store depletion can secondarily activate plasma membrane calcium channels, allowing an influx of calcium into the cytosol. Depletion of ER calcium stores leads to ER stress and ultimately leads to cell death (Sarco(endo)plasmatic reticulum Ca growing . One of  growing  and the pparatus . Thapsigll death .2+-pump(s) thereby blocking them  triggered the search for the cause of this similarity. Is, perhaps, thapsigargin toxic because it displaces a natural sesquiterpenoid ligand from its binding site on Caing them ? It is king them  are sesqhe pumps .2+ pumps  Ca2+ channels and acting as agonists of Ca2+ pumps that remove excess Ca2+. This made 2+ homeostasis system in its totality. Even ecdysteroids and hydrophobic vertebrate steroid hormones may act in a similar way.In combination, the picture emerges that farnesol/FLSs act on keeping intracellular Ca2+, and kinase C. This view gained a rather wide acceptance among molecular biologists, but not among cell biologists. The major objection is that, up to the present day, it has not been experimentally demonstrated that farnesol/JHs, which are highly hydrophobic molecules, ever enter the nucleus with its internal hydrophilic watery environment. As long as the in situ binding between JH and its Met-transcription factor inside the nucleus has not been experimentally proven, Met does not meet the required conditions to be classified as a “genuine receptor.” According to target. Even more, it is probable that Met is a Ca2+-sensitive target (2+ Pumping by Sarco(Endo)Plasmatic Reticulum Ca2+-Atpases And Endogenous Sesquiterpenoids”). To our knowledge, no data are available on nuclear receptors for farnesol in chordates. Farnesol/JH influences many other targets, most of them residing in membranes as cited before  and this in a developmental stage in which the CA are inactive. They named it “exocrine JH synthesis.” They also reported that this male accessory gland (MAG) JH was not secreted into the hemolymph, but instead left the male body along with secretory proteins that during copulation are transferred into the female’s genital system. This selectivity, or better unidirectionality, in JH secretion triggered Hyalophora. This way, the idea of “Golgicrine farnesol/FLS synthesis and secretion” was born. Immediately, the question emerged whether such “Golgicrine activity” is compatible with JH synthesis by the CA.The successive steps in this development have recently been reviewed by Leucophaea maderae. Similar results were later published by Acheta domesticus. Although it was known since long that Golgi systems serve a role in the secretion of proteins, the possibility that the “juvenilizing factor” secreted by the CA might be a protein was not considered at that time. The farnesol line of thinking , distributes itself between the membranes of the Golgi and hydrophobic stretches of amino acids present in the Golgi’s lumen where they can be further modified. Because both the membrane and the content of the lumen of the Golgi move toward the trans side where pinching off happens, farnesol/FLS also move along. We propose the term “lipid membrane way mode of transport” or “inbrome way of transport”  from its binding site on the SERCA pump. That ligand could be farnesol itself or a farnesol-like sesquiterpenoid (FLS) . As alrecis side. At the trans side, the opposite happens. The trans vesicles will, in their turn, fuse with the plasma membrane and discharge their content into the cell’s exterior. All these fusion and pinching off require instant closing of the membranes involved; otherwise, the electrical properties of the secreting cells would get severely impaired by the loss of inorganic ions, which are gradient-wise distributed over the membranes. By their nature, sesquiterpenoids are highly flexible molecules as indicated by their high rotatable bond count (7 according to PubChem) and horseshoe-shape form. In addition, they can be anchored in the lipid bilayer part of membranes as shown for the membrane attachment of Ras by A prominent feature of Golgi membranes is their high flexibility and capacity of self-closing. In active Golgi systems, vesicles that are pinched off from the RER will fuse with membranes of the Golgi at its In a former paper, The bigger an animal, the higher its membrane turnover. In insects, the CA in larvae may produce enough JH to lubricate the plasma membranes of all cells. This may be facilitated by the fact that the farnesol esters with JH activity  ensure that only very low amounts of JHs are needed to accommodate the whole body.H. cecropia. But even in the adult stage, when the CA are active, an additional supply of farnesol may be required, e.g., in the gonads with their numerous microvilli and secretory vesicles in some cell types in both males and females  are likely. Flowers are structures in which massive cell division takes place, an activity for which plasma membrane flexibility and turnover is, by definition, a major issue. Second, a function as “membrane liquidizer” or “membrane lubricant” is the last thing most researchers would think about. Yet, as we know from machine functioning, moving parts need to be lubricated. But this flexibility/lubricant problem is not restricted to animal cells only, but to all eukaryotic ones. That may be an explanation for the omnipresence of farnesol/FLS synthesis by the ubiquitous mevalonate biosynthetic pathway. Third, it is only recently that the key role of the mevalonate biosynthetic pathway in Ca picture .For a long time, farnesol was best known as a component of flower extracts to be used in the perfume industry. Its basic physiological role in plants is still poorly documented. In animal physiology, a possible role as a component of the “smell” of  animals has not yet been considered. In our opinion, there is no reason to assume that with respect to “the  perfume aspect,” the situation in animals should differ very much from that in plants.Only a few possible topics are listed:The key question: Is farnesol/FLS a hormone in vertebrates? Which stereoisomers do occur? Do SERCAs and SPCA pumps need a farnesol/FLS-like agonist? How would such agonists be attached: by hydrophobic interaction or by prenylation or still in another way? Do all tissues with active Golgi synthesize farnesol/FLS? Does the “inbrome model” of transport also apply to hydrophobic sex steroids?Identify the protein or peptide secreted by the Golgi present in the CA. Are enzymes involved in JH synthesis anchored in the membranes of the Golgi? Why does “exocrine JH” not diffuse into the hemolymph compartment? Does farnesol/FLS, due to its hydrophobic nature, ever end up in the nucleus? How is exocrine JH complementary to endocrine JH?Vachellia farnesiana that had been planted in the garden of Odoardo Farnese in Rome. To our knowledge, the exact physiological role of farnesol in plants is still unknown to date. But such ignorance about the functionality of farnesol/FLS also covers the great majority of all eukaryotes. They all synthesize farnesol. Even in vertebrates, early 2019 farnesol was named “a noble unknown”  gets fully inactivated with a drop of farnesol/FLS titers to zero as a result. The hormone secreted by the CA that, when absent, induced metamorphosis had been named “juvenile hormone.” 2+ channels  of JH in insects . It looks plausible to assume that the major mechanisms of their transport and mode of action at the level of the plasma membrane may be similar to those of farnesol/JHs.2+ homeostasis. Otherwise, they would have disappeared in some subdivisions of the eukaryotes, but they did not.The conclusions of this paper are based on a combination of experimental data and logical deduction. We fully agree with the objection that, for the moment being, too few experimental data are available to fully generalize “insect data” to ubiquitous mechanisms that we suppose operate in all eukaryotes. We nevertheless maintain our generalizations because we assume that the Golgi system has been well conserved in evolution for billions of years, along with the mevalonate biosynthetic pathway that is also omnipresent in eukaryotes because both serve an essential function, namely, in Ca2+ homeostasis and in farnesol/FLS production, are accepted, the novel paradigm says that it is normal and almost self-evident that all protein-secreting cells and tissues may produce their own FLSs. Whether there are quantitative and qualitative (types of isomers) differences remains to be investigated. The data that farnesyldiphosphate synthase is only abundant in the CA and not in any other tissue known to synthesize farnesol/FLS  was vigorously “defended” for over two decades against disagreeing researchers: no way to publish “aberrant” experimental data. Today’s situation is very different. If the two major functions of the Golgi, namely, roles in Caesol/FLS  suggest With this review, we intend to stimulate research in both basic and medical sciences into the full role of the Golgi. Malfunctioning of the Golgi system is implicated in several diseases, e.g., breast cancer and Haily–Haily disease . De LoofWe also intend to contribute to eliminate the goal-oriented thinking on evolution, which is still unconsciously propagated in textbooks: see the “milk production case” in this paper in which many people still mistake result for physiological necessity.Both authors jointly conceived and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acute myeloid leukemia (AML) is a common adult leukemia often arising from a preexistent myelodysplastic syndrome (MDS). High mortality rates of AML are caused by relapse and chemoresistance; therefore, we analyzed the role of P2X7 receptor (P2X7R) splice variants A and B in AML progression and response to chemotherapy. The expression of P2X7RA and P2X7RB was investigated in samples obtained from MDS and AML untreated subjects or AML patients in relapse or remission after chemotherapy. Both P2X7RA and P2X7RB were overexpressed in AML versus MDS suggesting a disease-promoting function. However, in relapsing patients, P2X7RA was downmodulated, while P2X7RB was upmodulated. Treatment with daunorubicin (DNR), one of the main chemotherapeutics for AML, upregulated P2X7RB expression while reducing P2X7RA mRNA in AML blasts. Interestingly, DNR administration also caused ATP release from AML blasts suggesting that, following chemotherapy, activation of the receptor isoforms via their agonist will be responsible for the differential survival of blasts overexpressing P2X7RA versus P2X7RB. Indeed, AML blasts expressing high levels of P2X7RA were more prone to cell death if exposed to DNR, while those overexpressing P2X7RB were more vital and even protected against DNR toxicity. These data were reproducible also in HEK-293 cells separately expressing P2X7RA and B. P2X7RA facilitation of DNR toxicity was in part due to increased uptake of the drug inside the cell that was lost upon P2X7RB expression. Finally, in an AML xenograft model administration of DNR or the P2X7R antagonist, AZ10606120 significantly reduced leukemic growth and coadministration of the drugs proved more efficacious than single treatment as it reduced both P2X7RA and P2X7RB levels and downmodulated c-myc oncogene. Taken together, our data suggest P2X7RA and P2X7RB as potential prognostic markers for AML and P2X7RB as a therapeutic target to overcome chemoresistance in AML relapsing patients. In some cases, AML can originate from an evolving MDS, i.e., a clonal disorder of hematopoietic stem cells2. Despite several efforts have been made to improve the clinical outcome of AML, current drugs fail to eliminate the leukemic stem cells responsible for the disease1. Therefore, the prognosis remains poor with most AML patients developing drug resistance. Accordingly, the identification of new progression/relapse biomarkers and potential therapeutic targets remains an urgent clinical goal in AML3.Acute myeloid leukemia (AML) is a clonal disorder characterized by the proliferation and the accumulation of myeloid precursors in the bone marrow (BM) that show aberrant differentiation patterns leading to hematopoietic impairment6. Haemopoietic lymphoproliferative disorders, among which AML, were the first neoplasias where P2X7R oncogenic activity was demonstrated9. However, evidence on the role of ATP and P2X7R in leukemia etiopathogenesis remains limited12.The P2X7 receptor (P2X7R) emerged as a promising therapeutic target in oncology, due to its widespread expression in cancer cells and the efficacy of its antagonist in reducing cancer growth and dissemination in animal models15. Formation of the macropore is dependent upon the presence of the long intracellular carboxyl tail as its deletion prevents pore formation while leaving unaltered the ion channel activity16. P2X7R activated macropore opening was also shown to enhance the intracellular uptake of drugs including chemotherapeutics such as doxorubicin17 and was therefore proposed as tumor cell-specific drug delivery system19. The structure of P2X7R C terminal was recently reported20 and it was speculated that this region was acquired by genomic rearrangement from a P2X4R like-gene in ancient jawed vertebrates generating the actual mammalian P2X7R21. It is therefore not surprising that certain species such as humans still express functional P2X7R splice variants missing the C terminal domain such as P2X7RB, an isoform unable to form the macropore, and thus without cytotoxic activity, but still endowed with ion channel properties23. In recent years, others and we demonstrated a growth-promoting activity for P2X7RB in cell lines24 and osteosarcoma24 as well as its involvement in metastasis and transformation26. The tumor-promoting activity of P2X7RB is shared by the full-length P2X7RA isoform accelerates cancer growth and dissemination and associates with poor prognosis in different malignancies29. However, to our knowledge, the effect of in vivo P2X7R blockade or chemotherapy on P2X7R isoforms expression was never tested and an extensive analysis of the distinct behavior of P2X7RA and P2X7RB in AML patients was never performed. The present study was aimed at identifying P2X7RA and B as novel biomarkers of AML response to chemotherapy and as new therapeutic targets for the disease. We covered P2X7R isoforms expression in an MDS and AML population and their behavior in vitro and in vivo models of the pathology, following chemotherapy and P2X7R antagonist administration.P2X7R is an ATP-gated ion channel that, when stimulated with high concentrations of agonists, triggers cell death via the opening of a large nonselective plasma membrane pore (macropore)TM Protease and Phosphatase Inhibitor Cocktail, EDTA-Free were acquired from Thermo Scientific.RPMI 1640, Iscove’s Modified Dulbecco’s Medium, nonessential amino acids, hygromycin, daunorubicin (DNR), and BzATP were from Sigma-Aldrich . DMEM high glucose medium, fetal bovine serum, penicillin, and streptomycin solutions were acquired from Euroclone . AZ10606120 was purchased from Tocris Bioscience . The reverse transcription kit, Real-Time PCR TaqMan reagents, Halt10, from the peripheral blood or BM of 68 patients subdivided according to diagnostic phase in 10 myelodysplastic syndrome (MDS), 47 first diagnosed of AML, 6 with relapse of the disease after chemotherapy, and 5 remitting AML patients. The main patients’ clinical characteristics are summarized in Supplementary Table Primary leukemic cells were used for mRNA extraction and obtained, as previously described23. A comparative CT experiment (ΔΔCT) was run to allow determination of fold increase of the target cDNA in the test sample relative to Te85 cell line reference sample, which was previously demonstrated to express minimal levels of both P2X7RA and B24. Customized TaqMan  primers and probes were for P2X7RA: forward primer: 5′ CGGCTCAACCCTCTCCTACT‐3′; reverse primer: 5′GGAGTAAGTGTCGATGAGGAAGTC 3′; 3′FAM probe: 5′ CACAGCGGCCAGACCG 3′. For P2X7RB, forward primer: 5′ GGAAAATGGTTTGGAGAAGGAAGTG 3′; reverse primer: 5′ CGATGAGGAAGTCGATGAACACA 3′; 3′FAM probe: 5′ ACAAGCGCTGCGTTAGT 3′. GAPDH reference was a Pre-Developed TaqMan Assay .Total RNA was extracted using the PureLink RNA Mini Kit (Thermo Scientific). Real-time quantitative PCR was performed as previously described23 and periodically tested for the absence of mycoplasma by MycoAlert kit (Euroclone). Culture media were RPMI 1640 medium plus nonessential amino acids (HL-60 and AML blasts) or DMEM high glucose (HEK-293), supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin.HL-60 human promyelocytic, HEK-293 stably expressing P2X7RA, P2X7RB, or empty vectors (MOCK) cell lines were previously available at Adinolfi laboratoryCell lysates or tumor homogenates were loaded in 4–12% NuPAGE Bis-Tris precast gels (Thermo Scientific) and proteins were separated and transferred onto a nitrocellulose blotting membrane . Membranes were incubated overnight at 4 °C with primary antibodies as follows: anti-P2X7  antibody, recognizing the C terminal domain of the protein was diluted 1:300, anti-P2X7  antibody, recognizing the extracellular loop of the protein was diluted 1:300, anti-c-myc antibody  was diluted 1:2500, and anti-SKP-1 antibody  was diluted 1:1000. Membranes were then incubated with secondary goat anti-rabbit  or goat anti-mouse , HRP-conjugated antibodies at a 1:3000 dilution. When required blocking peptide for the APR008 antibody  was added to the primary antibody at a 1:2 ratio and preincubated for 5 h before proceeding with the above-described immunostaining protocol. Protein bands were visualized by ECL HRP Chemiluminescent Substrate ETA C ULTRA 2.0  with a Licor C-Digit Model 3600. Densitometric analysis was carried out with ImageJ software and data were normalized on SKP-1 content.31.Measurement of intracellular calcium concentration with FURA-2AM and of plasma membrane permeabilization to ethidium bromide was performed as previously described12.ATP concentration was evaluated with ENLITEN rLuciferase/Luciferin reagent , according to the manufacturer’s instructions as previously describedAML blasts were resuspended in RPMI medium and treated with PBS or 200 nM DNR for 6 h. HEK cells seeded in serum-free medium let adhere and subsequently treated with PBS or 200 nM DNR for 48 h. Cell numbers were assessed at time 0 and 6 (blasts) or at time 0, 24, and 48 h (HEK). The Alamar blue assay was performed as per the manufacturer’s instructions after 24 h of incubation with either PBS or 200 nM DNR.4, 1 mM NaH2PO4, 20 mM HEPES, 5.5 mM glucose, 5 mM NaHCO3, and 1 mM CaCl2, pH 7.4) plus 1 µg/ml DNR and whenever required 3 mM ATP. Fluorescence was captured at 328 nm excitation and 545 nm emission.HEK MOCK, P2X7RA, and P2X7RB were imaged with a Nikon Eclipse TE300 inverted microscope , while HIGH-P2X7RA and HIGH-P2X7RB AML blasts with a Leica DMI 4000B (Leica Microsystems CMS GmbH) microscope in saline solution  to receive intramass injections of sterile PBS (placebo), DNR (150 µg/ml), AZ10606120 (2 μM), or both compounds at post-inoculum days 8 and 10 as previously described33. The operator was blinded to the group of allocation. Tumor size was measured at post-inoculum days 8, 10, and 12 and ex vivo with a caliper and volume calculated according to the following equation: volume = π/6[w1 × (w2)2], where w1 = major diameter and w2 = minor diameter. Mice were euthanized 12 days after cell inoculum and excised tumors were homogenized and processed for further analysis.Animal procedures were approved by the University of Ferrara ethic committee and the Italian Ministry of Health. In vivo experiments were performed in the athymic nude-Foxn1nu strain acquired from Envigo . A sample size of 12 animals per condition was computed a priori with the G*power softwaret test performed with the GraphPad Prism software . Coding: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.All data are shown as mean ± standard error of the mean (SEM). Except for real-time PCR data reported in Fig. 10, however the analysis of the differential expression of P2X7RA and B isoforms in the disease was missing. Therefore, we investigated by Real time PCR mRNA levels of P2X7RA and B in 58 patients affected with AML and 10 patients affected with MDS. AML patients were further subdivided according to the diagnostic phase and response to treatment into three groups: (1) newly diagnosed untreated subjects , (2) relapsing subjects, with a return of the pathology after chemotherapy , or (3) remitting patients, with no evident reappearance of the pathology one to three months after chemotherapy . mRNA levels of P2X7RA and B of AML de novo patients were massively increased as compared to MDS blasts, supporting the hypothesis that both receptor variants positively correlate with disease progression , this class of drugs is known to cause an increase of ATP in the TMEsts Fig.  and, intsts Fig. . When ansts Fig.  from thosts Fig. . Interessts Fig. . To furtath Fig. . These dath Fig.  and vitaath Fig. . DNR treath Fig.  with a s14, therefore we tested whether P2X7RA can further facilitate DNR-dependent cell death by favoring its cellular uptake. To test this hypothesis we took advantage of the natural red fluorescence emitted by DNR to measure its loading inside cells treated with 3 mM ATP (Fig. P2X7RA-mediated pore opening is known to mediate cell permeabilization to large moleculesATP Fig. . SimilarATP Fig. .Fig. 4P235, therefore we asked whether AZ10606120 could similarly affect the expression of c-myc that is a well-known oncogene in AML and MDS36. Figure To analyze the role of P2X7R isoforms in an AML in vivo model, we took advantage of HL-60 human promyelocytic cell line that expresses both P2X7RA and B at the mRNA and protein level Fig. , shows f37 and its targeting reduced leukemic growth in AML experimental models12. In vitro experiments also showed that high doses of ATP selectively disrupted the growth of leukemic blasts versus normal haemopoietic stem cells10. However, these studies focused on P2X7RA isoform that is known to mediate both cell proliferation upon stimulation by low doses of agonist and cell death through the opening of a large unselective pore in the presence of millimolar concentrations of ATP38. Humans also express another splice variant of the receptor named P2X7RB22, which while retaining the proliferation-promoting activity loses the ability to mediate the opening of the cytotoxic pore23. Limited evidence is available on the physio-pathological role of P2X7RB, we know that it is expressed mainly in the central and peripheral nervous system and in leukemic cells23 and that, its expression associates to stem cell differentiation39. In solid cancers, P2X7RB was hypothesized to participate in proliferation24, matrix invasion,26 and tumor spreading25. To better understand the different roles of P2X7RA and P2X7RB splice variants in leukemias, we investigated the expression of these isoforms in patients affected by MDS or AML and in AML relapsing and remitting patients. Both P2X7RA and P2X7RB mRNA levels were strongly increased in firstly diagnosed AML as compared to MDS, supporting the hypothesis that both receptor variants positively correlate with disease progression. Interestingly, AML relapsing patients after chemotherapy were characterized by differential expression of P2X7RA and P2X7RB as compared to de novo patients as while P2X7RA expression was significantly reduced, P2X7RB expression significantly increased. On the contrary, in remitting AML patients both P2X7RA and B expression was significantly decreased. These data suggest that chemotherapy may cause a decrease in P2X7RA expression while positively modulating P2X7RB and therefore that individuals expressing high levels of P2X7RB could be resistant to chemotherapy, prone to relapse and refractory to treatment.The P2X7R was previously reported to be expressed and functional in AML11 and confirmed these data in all cell types tested in the present study. Based on these findings, we hypothesized that chemotherapy could cause a release of ATP from dying leukemic cells, which will, on one hand, kill P2X7RA overexpressing blasts through macropore formation and, on the other facilitate the proliferation of P2X7RB expressing blasts that do not undergo cell death but on the contrary exploit ATP-dependent proliferation (see Fig. 17. Indeed, this proved to be the case as DNR uptake by both HEK P2X7RA and HIGH-P2X7RA AML blasts stimulated with 3 mM ATP was higher than in HEK mock controls, HEK P2X7RB cells, and HIGH-P2X7RB AML blasts, respectively.We recently demonstrated that the chemotherapeutic DNR can cause an increase of ATP in the leukemic microenvironment35, and suggest a mechanism for P2X7R-mediated transformation in AML. c-myc is commonly overexpressed in both AML and MDS40 as it is a central transcription factor in the maturation of hematopoietic stem cells, thus exerting a critical function in hematopoietic malignancies41. Moreover, c-myc is involved in AML cells resistance to chemotherapy and therefore identifying new drugs able to downmodulate c-myc levels might greatly improve the therapeutic approach of AML relapsing patients43. AML relapse is the main cause of patients’ death and efforts to improve their survival is an important goal for onco-hematologists to ensure patients’ recovery. Our study suggests that P2X7R isoforms could be exploited as valuable prognostic markers to predict response to chemotherapy. Moreover, P2X7R could be an interesting target for a new combined therapy in AML. Based on our data, we suggest a therapeutic approach with DNR administered as first-line, to eliminate P2X7RA-expressing leukemic cells. P2X7R antagonists could be administered in a second phase to those that developed chemotherapy resistance and whose blasts are positive for P2X7RB expression. Of interest, several P2X7R targeting drugs are under clinical trials for other pathologies and proved to be well tolerated with limited side effects thus favoring their use also in oncologic patients45.To validate patients’ data in an in vivo model of leukemia we took advantage of HL-60 human promyelocytic cell line, which expresses both P2X7R isoforms, can form the cytolytic pore and releases ATP upon DNR stimulation. HL-60-derived leukemia-bearing mice were treated with DNR and with a P2X7R antagonist, alone or in coadministration. Both compounds significantly reduced leukemia growth, but their combination was more efficient than single-drug treatment. DNR effect on P2X7R isoforms expression was similar to what was seen in relapsing patients as P2X7RB expression was significantly increased by DNR, while P2X7RA showed a tendency to decrease. Interestingly, coadministration of DNR with P2X7R antagonist normalized both P2X7RA and B levels and reduced intratumor levels of the c-myc oncogene. These data are in line with our previous demonstration that P2X7R blockade decreases expression of the N-myc oncogene, another member of the myc familysupplemental table 1supplementary table legendsupplemental figure 1supplementary figure legend


Estrogen receptor (ER) signaling has been widely studied in a variety of solid tumors, where the differential expression of ERα and ERβ subtypes can impact prognosis. ER signaling has only recently emerged as a target of interest in acute myeloid leukemia (AML), an aggressive hematological malignancy with sub-optimal therapeutic options and poor clinical outcomes. In a variety of tumors, ERα activation has proliferative effects, while ERβ targeting results in cell senescence or death. Aberrant ER expression and hypermethylation have been characterized in AML, making ER targeting in this disease of great interest. This review describes the expression patterns of ERα and ERβ in AML and discusses the differing signaling pathways associated with each of these receptors. Furthermore, we assess how these signaling pathways can be targeted by various selective estrogen receptor modulators to induce AML cell death. We also provide insight into ER targeting in AML and discuss pending questions that require further study. Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, accounting for approximately 21,000 new diagnoses annually in the U.S. . SimilarAML can result from an accumulation of genetic mutations, chromosomal translocations and/or epigenetic modifications that occur in numerous combinations and are unique from patient to patient . This vaEstrogen signaling occurs through two estrogen receptors (ERs): ERα and ERβ, which are encoded by the ESR1 and ESR2 genes, respectively . AlthougAlthough AML is not a sex-hormone-related disease, interest in the possible involvement of estrogen and estrogen receptors in AML has stemmed from several epidemiological studies showing sex differences in the incidence of hematological malignancies. Men are approximately two times more likely to be diagnosed with acute or chronic lymphocytic leukemia and various lymphomas, suggesting that estrogen may play a protective role in the development of these diseases ,22. Diff−/−) but not in ESR2 (ESR2−/−) knock-out mice [Estrogen signaling has emerged as an important factor in hematopoiesis within the last decade. Sex differences in HSCs show a direct role of estrogen in HSC maintenance and growth. In ovariectomized female rats, the number of HSCs and colony forming units of bone marrow cells are significantly reduced, which suggests that estrogen is required for normal hematopoiesis . Similarout mice . Indeed,out mice ,27,28. Nout mice ,29. By cout mice . In one out mice . Both ERout mice ,33,34. Iout mice ,36. Addiout mice ,37. GATAout mice . By contout mice .These studies, summarized in ERα is expressed in a subset of patient-derived AML cells ,40. The −/−) resulted in hypercellularity of the bone marrow and myeloproliferative neoplasia resembling chronic myeloid leukemia  -/- leukemia cells should be conducted to confirm this hypothesis. In another study, tamoxifen and the sphingolipid, C6-ceramide, synergistically induced apoptosis in AML cells and primary samples while sparing normal peripheral blood mononuclear cells (PBMCs) [Tamoxifen is a SERM with tissue-specific agonist and antagonist activities. Tamoxifen is an ERα antagonist and ERβ agonist in the breast where its antiestrogenic activity is used to treat ERα-positive breast cancer . By cont (PBMCs) . The com (PBMCs) . Tamoxif (PBMCs)  through  (PBMCs) . These fDiosmetin is a member of the phytoestrogens, which are secondary plant metabolites that are structurally and functionally similar to mammalian estrogen and thus exert estrogenic activity . The antDiosmetin inhibited the primary and secondary engraftment of patient-derived AML cells with no effect on normal CD34+ cell engraftment . ERβ actGenistein is a phytoestrogen found mainly in soy . A commoQuercetin is a flavanol with estrogenic activity that naturally occurs in many fruits and vegetables . SimilarAML is an aggressive disease that requires new drug targets and therapeutic options. Although not regarded as a sex-hormone-related disease, epidemiological and preclinical studies provide compelling data to support ER targeting in AML. Studies have shown that ERα activation enhances the efficacy of conventional chemotherapeutics and that ERβ suppresses leukemogenesis and reduces leukemia cell growth.In considering ER targeting as an anti-leukemia strategy, it is imperative that the ERβ/ERα ratio in the tested population is known and that the binding of the ER modulator is well defined. Since ERα and ERβ exert opposing actions, ideal drug candidates should be subtype-specific. Furthermore, current evidence provides greater support for specific ERβ agonists as ideal drug candidates, given their anti-proliferative effects in several solid tumors and in AML. Phytoestrogens are of great interest in targeting ERβ, as many of them have demonstrated preferential binding to ERβ over ERα . SynthetMechanistic studies show that ER modulators activate apoptotic mechanisms, cause mitochondrial dysfunction, or modulate protein kinase signaling and result in cell death. While most studies are correlative, few have demonstrated the functional importance of ER signaling to mitochondrial dysfunction and apoptosis ,77. Give