It is not understood why healthy tissues can exhibit varying levels of sensitivity to the same toxic stimuli. Using BH3 profiling, we find that mitochondria of many adult somatic tissues, including brain, heart, and kidneys, are profoundly refractory to pro-apoptotic signaling, leading to cellular resistance to cytotoxic chemotherapies and ionizing radiation. In contrast, mitochondria from these tissues in young mice and humans are primed for apoptosis, predisposing them to undergo cell death in response to genotoxic damage. While expression of the apoptotic protein machinery is nearly absent by adulthood, in young tissues its expression is driven by c-Myc, linking developmental growth to cell death. These differences may explain why pediatric cancer patients have a higher risk of developing treatment-associated toxicities.

Pediatric cancer patients treated with ionizing radiation or cytotoxic chemotherapy have a high risk of developing devastating toxicities including cognitive decline and chronic heart failure, limiting the use of potentially curative therapies. These treatments, which rely on the preferential induction of an apoptotic cell death in cancer cells over healthy tissues, are comparatively well tolerated in adults, yet the molecular basis for this difference in sensitivity is unknown. Herein, we make the discovery that apoptosis is dynamically regulated during post-natal development in healthy tissues, altering cell fate in response to genotoxic damage induced by anti-cancer therapies. Importantly, we use mouse models to show that these pathways may be modulated to potentially prevent treatment-associated toxicities.

Much of the study of apoptosis has been devoted to cancer and hematopoietic tissues, with relatively little study of other healthy somatic tissues. It is currently unknown whether cells that make up distinct tissues have varying levels of apoptotic priming, which could potentially contribute to their different sensitivities to classical apoptosis-inducing agents.

Chemotherapy and radiation treatments have cured cancer in millions of patients (), yet the apoptotic cell death that these agents can induce in healthy tissues limits their use. This is especially true in pediatric patients who experience considerably higher levels of treatment-associated toxicity and morbidity from genotoxic agents. For example, brain irradiation is a critical component of the potentially curative treatment for brain tumors. However, radiation can also trigger cell death in healthy neurons in very young patients, resulting in permanent and devastating cognitive deficits with severity being inversely correlated with age (). Similarly, children with many types of cancers are commonly treated with anthracyclines including doxorubicin. However, doxorubicin treatment in children can cause thinning of the cardiac ventricular walls, a reduction in ventricular mass, and consequent heart failure, with the youngest children again being most at risk (). Currently, parents and clinicians must balance the curative potential of these treatments with their potential for causing devastating toxicities. It is unclear why young children are more at risk of developing these toxicities than adults.

The mitochondrial apoptosis pathway can be activated by a wide variety of cellular stressors including growth factor or nutrient deprivation as well as genotoxic damage from cytotoxic chemotherapies and radiation. In each of these cases, the basal state of the mitochondrial apoptotic pathway can alter the eventual fate of the cell (). To directly measure the functional state of the mitochondrial apoptotic pathway in cells, we developed the BH3 profiling assay, which measures apoptotic priming (proximity of cellular mitochondria to the apoptotic threshold) by delivering titrated doses of distinct pro-apoptotic signals (BH3 peptides) to mitochondria while monitoring MOMP (). In this assay, mitochondria bearing only a small reserve of unbound anti-apoptotic proteins undergo MOMP in response to even relatively low doses of pro-apoptotic peptides and are thus classified as “primed” for apoptosis; primed cells readily die when pro-death signals are generated in response to cellular damage or stress (). In contrast, cells that contain a large reserve of unbound anti-apoptotic proteins are less sensitive to BH3 peptides and are classified as “unprimed”; unprimed cells must experience higher levels of damage or stress to trigger MOMP. Finally, cells that block apoptosis by not expressing sufficient levels of critical components of the cell death machinery (such as BAX and BAK) are classified as “apoptosis refractory.” We have shown previously that patients with primed cancers respond more favorably to chemotherapy than patients with unprimed cancers ().

The intrinsic, or mitochondrial, pathway of apoptosis is an evolutionarily conserved and highly regulated form of cell death that is critical for development and homeostasis of multicellular organisms. The deregulation of apoptosis is associated with many pathologies including cancer (). Apoptosis is triggered when a pro-apoptotic effector protein (BAX or BAK) is activated by an activator BH3-only protein, of which BIM and BID are most potent (). This results in the oligomerization of BAX or BAK, causing mitochondrial outer membrane permeabilization (MOMP) and consequent release of cytochrome c into the cytosol, where it complexes with APAF-1 to form the apoptosome. This complex activates downstream cysteine proteases, including caspase-3, that dismantle the cell and promote phagocytosis (). However, anti-apoptotic proteins in this family (BCL-2, BCL-X, MCL-1, etc.) can block apoptosis by binding and sequestering monomeric BAX/BAK or BH3-only proteins (). In order for apoptosis to occur, anti-apoptotic proteins within the cell must be overwhelmed and BAX and/or BAK activated.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

The dynamic regulation of apoptosis in murine tissues dramatically affected responses to genotoxic damage and prompted us to ask whether the same temporal regulation could be directly observed in human tissues. One source of non-malignant, viable human brain tissue is from pediatric and adult patients who undergo removal of seizure foci to control otherwise intractable seizures caused by epilepsy, trauma, or other pathology. Seizure foci are mapped in the human brain and resected, inevitably along with some ostensibly healthy brain tissue (). We obtained fresh, healthy brain tissue from over 20 such patients from 4 months to 21 years of age for BH3 profiling analysis ( Table S3 ). As with mice, we found human brain tissue from young patients to be significantly more sensitive to BIM and BID BH3 peptides than those from adults ( Figures 7 H and 7I). There existed a consistent downregulation of apoptotic priming during post-natal human brain development, predisposing the youngest children to radiation- and chemotherapy-induced neurotoxicity. In our study, there was a period of higher heterogeneity in apoptotic priming among patients between 2 and 6 years old, after which the brain transitions to full apoptotic resistance. Finally, we mined mRNA expression data in the human brain during pre- and post-natal development () and found that BAX expression is dramatically reduced during development, starting even prenatally ( Figure 7 I), which we confirmed at the protein level in our samples ( Figure 7 J). The highest mRNA expression of BAX was found in fetal brain, suggesting that the highest levels of apoptotic priming, perhaps similar to those observed in the youngest (P0–P2) mice, would be found in fetuses and pre-term infants.

Because modulation of Myc was sufficient to alter priming in the expected manner, we next sought to test whether Myc directly drives the expression of apoptosis-regulating genes in neonatal tissues. Myc activates the transcription of its target genes by binding specific E box (enhancer box) elements in gene promoters (). We therefore used chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) to test for Myc occupancy in the E boxes present in the promoters of various BCL-2 family member genes. In agreement with our hypothesis that Myc may be modulating BAX expression in neonatal tissues, we detected the Bax promoter being bound by Myc in neonatal brain, kidney, liver, and spleen tissues ( Figure 7 G). Furthermore, Myc binding to the Bax promoter was lost in the adult brain and kidney but not the spleen, consistent with the spleen maintaining a high level of priming throughout life (note that adult heart and liver tissues were not tested). We also detected E boxes in the promoters of pro-apoptotic Bcl2l11 (Bim), which is consistent with previous reports (), and Bid, and confirmed that Myc bound to these promoters. Taken together, these data indicate that Myc drives an apoptotically primed state by directly activating transcription of pro-apoptotic genes Bax, Bim, and Bid.

After acute liver damage, hepatocytes are able to re-enter the cell cycle and proliferate in order to replace dead cells (). Based on our previous data, we hypothesized that hepatocytes from adult liver tissue that are actively expressing Myc and proliferating would become more primed for apoptosis. We tested this by inducing immune-mediated liver damage via injection of concanavalin A (ConA), which triggers active liver regeneration and hepatocyte proliferation (). After injection with ConA, we observed a profound increase in Myc-positive cells within the liver (∼0% Myc-positive in untreated animals) ( Figure 7 E), as hepatocytes proliferated to replace dead cells. We directly compared the level of priming in Myc-positive versus Myc-negative hepatocytes and found the former to be significantly more sensitive to BIM and BID BH3 peptides ( Figure 7 F). Thus, using both genetic and physiologic models, we found that activation of Myc increases mitochondrial priming.

Next, we sought to determine whether activating Myc in adult tissues would be sufficient to reactivate apoptosis in refractory cells. We first utilized the well-characterized Gt(ROSA)26Sor(hereafter MycER) mouse model that was developed to study the physiological effects of acute Myc activation in adult tissues (). These mice express a cDNA encoding human c-Myc fused at its C terminus to the hormone-binding domain of 4-hydroxytamoxifen-responsive mutant murine estrogen receptor, enabling us to activate Myc systemically with injections of tamoxifen. Activation of Myc in normally apoptosis-refractory brain and kidney tissue, or unprimed liver tissue, increased sensitivity to genotoxic damage ( Figure 7 C). Notably, we found that Myc-positive cells within these tissues were more primed for apoptosis than their Myc-negative counterparts as measured via flow cytometry-based BH3 profiling ( Figure 7 D). Our data suggest that Myc activation may re-enable the apoptotic pathway in apoptosis-refractory tissues.

Expression of Myc was associated with higher apoptotic priming, but this relationship may not be causal, prompting us to test whether Myc expression was necessary to maintain high priming in neonatal tissues. We therefore measured priming in the cerebral cortex of either WT or Mycmice (). As expected, the loss of one allele of Myc resulted in reduced sensitivity to BIM compared with WT littermates ( Figure 7 B).

Consistent with our hypothesis, we found that Myc is expressed at higher levels in young brain, heart, and kidney tissues compared with adult tissues ( Figure 4 A). The human proteome database did not contain sufficient data for Myc expression. If Myc was driving expression of pro-apoptotic genes, we would expect Myc-expressing cells to be more primed. To test this, we utilized flow cytometry-based BH3 profiling, which allows for single-cell measurements of priming concurrently with evaluation of extracellular or intracellular factors. We measured priming and nuclear Myc expression (indicative of activation) in cells from P0 tissues and found that Myc-positive brain, kidney, and liver cells were, as expected, more primed than Myc-negative cells ( Figure 7 A ). We did not detect any Myc-positive cells within the respective adult tissues, which we again found to be dramatically less primed than neonatal tissues. In heart tissues, the extended processing necessary for this analysis resulted in prohibitively high levels of cytochrome c loss in even untreated cells, thus preventing analysis in that tissue.

(J) Immunoblotting for BAX across healthy human brain specimens tested in (H) and (I). Numbers indicate months post-conception. Heatmap represents expression of BAX normalized to GAPDH levels.

(I) Summary of fluorescence-based BH3 profiling data from human brain tissues. Each point represents an average of three measurements in each human brain specimen across at least five IEs. BAX mRNA expression across human brain regions were assessed at indicated ages.

(G) ChIP-qPCR was performed to measure Myc occupancy on promoters for positive control (Mybbp1a) and negative control (Untr6) genes, along with genes of interest. Values shown are binding events detected per μg chromatin. In (A), (B), (C), (D), (E), (F), and (G), bars represent means.

(F) 96 hr after injection with concanavalin A, liver tissue was collected and flow cytometry-based BH3 profiling was performed on hepatocytes while monitoring nuclear Myc expression. Each point represents one animal across three IEs.

(E) After injection with vehicle or concanavalin A, liver tissue was collected from mice at the indicated time points and Myc positivity was measured in hepatocytes via flow cytometry. Each point represents one animal across two IEs.

(D) Flow cytometry-based BH3 profiling was performed on tissues from (C) and each point represents the loss of cytochrome c in cells that are nuclear Myc-positive versus Myc-negative from each tamoxifen-treated animal across two IEs.

(C) MycER mice were treated with vehicle (oil) or tamoxifen (tam) to activate Myc for 3 days and then irradiated (8 Gy). TUNEL + cells were counted per 20× field.

(B) Fluorescence-based BH3 profiling was performed on brain tissue (neocortex) of litter-matched, neonatal Myc WT or heterozygous mice. Each point represents analysis of a single animal across two IEs.

(A) Flow cytometry-based BH3 profiling was performed on indicated tissues collected from neonatal or adult mice and loss of cytochrome c (indicative of apoptosis) was measured after treatment with negative control (DMSO), positive control alamethicin (AlaM), or BH3 peptides. For neonatal tissues, each point represents a flow cytometry-based measurement of either Myc-negative or Myc-positive cells (as determined by co-staining for nuclear Myc) from a single tissue sample. For adult tissues, each point represents a flow cytometry-based measurement of Myc-negative cells. All cells analyzed by flow cytometry were CD45 negative to exclude blood cells from analysis. Data shown are for either three (neonatal) or two (adult) IEs.

The concerted loss of several genes responsible for regulating apoptosis suggested that a master developmental program or transcription factor may be regulating multiple members of this pathway. One such potential modulator is c-Myc (hereafter Myc), a transcription factor that drives cellular growth and proliferation in normal as well as cancerous cells (). Several links between Myc and apoptosis have been reported, including the sensitization of Myc overexpressing cells to a variety of pro-apoptotic stimuli () and its direct regulation of BAX expression (). We thus hypothesized that tissues early in development express Myc at levels that are sufficient to drive the expression of BAX and potentially other apoptosis-related genes, making them primed for apoptosis, and that loss of BAX expression in adulthood is related to loss of Myc.

We next asked if suppression of the intrinsic apoptotic pathway could reverse these effects of doxorubicin. Using knockout mice to model pharmacologic inhibition, we found that loss of both Bax and Bak1 was required to consistently reverse doxorubicin-induced damage in neonatal mouse hearts ( Figure 6 E). In addition, knockout of Bax and Bak1 in days P5 or P6 mice treated with doxorubicin showed dramatically reduced, although not completely lost, evidence of early injury in MTS histology ( Figure S5 B). Therefore, young heart tissue expresses both BAX and BAK at sufficient levels to activate apoptosis, and thus inhibition of both of these effectors is needed to mitigate doxorubicin-induced thinning of ventricular walls and reduction in ventricular mass. In addition, there may also be a non-apoptotic component of doxorubicin-induced cardiotoxicity since the BAX/BAK double knockout mice still exhibited some signs of early injury based on MTS histology, although symptoms were greatly reduced. Taken together, our results show that hearts in young mice exhibit more severe clinically relevant symptoms of doxorubicin cardiotoxicity than adults, potentially due to the increased BAX and BAK expression in apoptotically primed young hearts ( Figure 2 B) that renders them hypersensitive to doxorubicin ( Figure 2 C).

We developed a mouse model of doxorubicin-induced cardiotoxicity by injecting mice with three doses of doxorubicin (5 mg/kg) over the course of 1 week, starting at days P5 or P6 (primed for apoptosis), P11 or P12 (unprimed), or P60-P80 (apoptosis refractory) ( Figure 6 A ). Our dosing schedule is reduced in intensity (three doses instead of five) from one previously used to model chronic doxorubicin cardiotoxicity in adult mice (), which, when tested in young mice, induced prohibitively high levels of cardiotoxicity as demonstrated by arrhythmias and death (data not shown). Using echocardiograms at day 14, we found significant thinning of the interventricular septal (IVS;d) and posterior ventricular walls (LVPW;d) in M-mode echocardiogram tracings at diastole in animals that began receiving injections at days P5 or P6, but not those that began as adults ( Figures 6 B–6D, Movies S1 and S2 ). Moreover, animals treated at a young age exhibited a profound decrease in EF and left ventricular mass while adults did not. Masson's Trichrome staining (MTS) of hearts after treatment showed focal areas of altered myocyte architecture with signs of early injury (loss of cross-striations, disorganization of myofilaments resulting in abnormal staining, and hypochromatic nuclei) in mice treated starting at days P5 or P6 but not adults ( Figure S5 A).

(D) Summary echocardiogram data. Each point represents an average of two measurements in each animal across five or more IEs. Bars represent means.

(C) Representative parasternal long-axis views at the level of the papillary muscle are shown from WT mice treated at day P5.

(A) Schematic representation of the experiment. WT mice of different ages were injected intraperitoneally with three doses of doxorubicin at 5 mg/kg on days 0, 4, and 7. Echocardiograms to assess heart function were performed on day 14.

Young hearts are considerably more sensitive to doxorubicin than non-elderly adult hearts. Clinically, the degree of cardiotoxicity observed correlates with age, with the youngest children being most at risk for developing symptoms including decreased ejection fraction (EF), thinning of ventricular walls, and an overall reduction in heart size (ventricular mass), sometimes referred to as “Grinch syndrome” (). We hypothesized that the differences in apoptotic priming in cardiomyocytes from young versus adult hearts may contribute to the heightened risk of developing cardiotoxicity in young patients.

We then found that each tissue exhibits its own pattern of effector dependence. Splenocytes can execute apoptosis via BAX or BAK and, although loss of BAX is somewhat protective, both must be lost in order to prevent all radiation-induced apoptosis ( Figure 5 C). In the neonatal heart, loss of either BAX or BAK reduced responses to peptides and radiation, indicating that both effectors are present, yet at limited levels, consistent with our immunoblotting results ( Figure 4 A). Cells within the neonatal kidney also contain both BAX and BAK, and loss of either reduced peptide responses. However, loss of BAX meaningfully reduced caspase activation after irradiation while loss of BAK did not. Finally, we found that neonatal hepatocytes contained both BAX and BAK and required the loss of both in order to significantly reduce apoptosis post-radiation. In all cases, the knockout of Bax preferentially dulled responses to the BIM BH3 peptide while loss of Bak1 preferentially dulled responses to the BID BH3 peptide, which is in agreement with reported specificity of activator/effector interactions (). These results may enable the prevention of damage-induced apoptosis in neonatal tissues by inhibiting BAX alone (brain) or both BAX and BAK (heart, kidney, liver, spleen).

BAX and BAK have non-overlapping roles in regulating apoptosis and their activity can be modulated selectively (). Our results showed that BAX and BAK are downregulated in tissue-specific manners as they transition from being apoptotically primed to refractory. We therefore utilized mouse models to determine the distinct contributions of BAX and BAK to the activation of apoptosis in healthy tissues. Neonatal brain tissue in wild-type (WT) mice was efficiently depolarized by the BIM (∼80%) and, to a lesser extent, the BID (40%) peptides ( Figures 5 A, 5B, and S4 ). In Baxneonates, however, we found the responses to BIM and BID significantly reduced, which was not evident in Bak1mice. We therefore hypothesized that loss of Bax would protect brain tissue from radiation-induced apoptosis while loss of Bak1 would not. In agreement with the BH3 profiling data, we found that loss of Bax prevented nearly all caspase-3 activation post-radiation while loss of Bak1 had no effect ( Figure 5 C). Thus, in the early post-natal brain, BAX and not BAK is the dominant effector that is engaged to trigger apoptosis.

(C) Summary caspase-3 activity data from P0 to P2 mouse tissues after whole-body irradiation. Each point represents an average of three measurements in each tissue across 11 IEs. Bars represent means.

(B) Summary BH3 profiling data from P0 to P2 mouse tissues. Each point represents an average of three measurements in each tissue across four IEs. Bars represent means.

(A) Tissues were collected from P0 mice of indicated genotypes and BH3 profiled. Representative traces are shown (mean ± SD, three or more IEs).

We also found BAX and BAK, along with other key components of the apoptotic machinery, to be strongly downregulated in human adult brain, heart and liver tissues relative to fetal tissues ( Figure 4 B and Tables S1 and S2 ).

Similar results to the brain were also observed in heart, kidney, and liver with BAX and BAK being rapidly downregulated following birth ( Figure 4 A). We confirmed that this downregulation occurs in the non-blood cells that comprise these tissues via IHC ( Figure S3 ). We also found that adult liver tissue expresses low, yet detectable, levels of BAK ( Figure S3 B), which is consistent with the higher sensitivity of adult hepatocytes to BID over BIM ( Figure 1 B) due to their activation preferences (). It is notable that anti-apoptotic proteins tended to be downregulated in these tissues as well, further demonstrating that adult tissues are not protected from apoptosis by high expression of anti-apoptotic proteins. Instead, there appears to be a wholesale dismantling of the apoptotic machinery to render these cells apoptosis refractory, preserving their survival. The expression levels of voltage-dependent anion channel, which can facilitate cytochrome c release from mitochondria during MOMP, as well as inhibitor of apoptosis proteins XIAP and CIAP1 remained largely unchanged across all tissues.

In contrast to the spleen, we found the expression of BCL-2 family members in the mouse brain during post-natal development to be dynamic. BAX was highly expressed in the mouse brain at P0–P5, but continually reduced into adulthood ( Figure 4 A). BAK was reduced in a similar manner yet expression levels were lower overall (relative to adult spleen). BCL-XL, as well as caspase-8 and caspase-9 were consistently expressed while BIM, BID, MCL-1, and BCL-2 were also reduced during post-natal brain development. Finally, both caspase-3 and APAF-1 were strongly downregulated during post-natal development, further contributing to the suppression of apoptosis in adult brain tissue.

The contrast in apoptotic priming in vital tissues between young and adult mice prompted us to explore the age-related expression of BCL-2 family proteins. Because we had shown previously that exogenous BAX supplementation is sufficient to reverse mitochondrial resistance to pro-apoptotic signals, we focused on the developmental regulation of BAX and BAK. In the spleen, we found the critical effectors BAX and BAK and potent activator BH3-only proteins BIM and BID to be consistently expressed during the entire lifespan of the animals ( Figure 4 A ). Major anti-apoptotic proteins were also expressed in the spleen consistently throughout life, although a downregulation of BCL-XL and MCL-1 and upregulation of BCL-2 with age was noted. Expression of caspase-3 and APAF-1 was unchanged over time. These results were consistent with BH3 profiling data showing high levels of priming throughout life.

Finally, we sought to determine whether the changes in apoptotic priming were dependent on an in vivo milieu or whether they could be driven by cell-autonomous mechanisms. To test this, we isolated hippocampal neurons from embryonic day 19 (E19) rat brains and measured the apoptotic priming and chemosensitivity of these cells at regular intervals as they matured in vitro ( Figures 3 A–3C ). These non-proliferating ( Figure 3 D) neurons underwent the same developmentally defined transition from high apoptotic priming to apoptotic resistance that was evident in vivo. Their sensitivity to the classical apoptosis inducers staurosporine and doxorubicin followed a similar pattern ( Figure 3 E).

(E) Chemosensitivity of rat hippocampal neurons in vitro. Each point represents an average of three measurements at each time point across two IEs.

(B) Primary neurons were BH3 profiled at indicated days in vitro. Representative traces are shown (mean ± SD, two IEs).

(A) Primary neurons were isolated from E19 rat embryos and allowed to mature in vitro, with representative images shown at indicated time points (days in vitro). Note that the same cells (those inside box on low magnification) are shown across the four time points in the higher magnification window. Scale bars, 200 μm.

Very young patients suffer increased iatrogenic cardiotoxicity from anthracyclines compared with non-elderly adults. To determine whether the dynamic levels of apoptotic priming during post-natal development may contribute to this difference, we injected mice at various post-natal stages with the anthracycline doxorubicin and measured caspase activation in the heart, again modeling pediatric cancer treatment in humans. In neonatal heart tissue, we detected caspase activation, which decreases with age in a manner similar to that following radiation damage ( Figure 2 C).

We next sought to test the functional consequences of these developmental changes in apoptotic priming. We chose to again stress cells with gamma radiation, which applies an equal and reproducible level of genotoxic damage across tissues and models radiation therapy in pediatric cancer patients. Mice at various stages of post-natal development were treated with whole-body gamma irradiation and apoptosis was quantified. We detected extensive caspase-3/-7 activation post-radiation, at doses of 0.50–8 Gy, in brain, heart, and kidney tissues in P0–P2 mice, which was measurably reduced each day of post-natal development and finally silenced (insensitivity to 8 Gy) by P13, P15, and P12 in brain, heart, and kidney, respectively ( Figures 2 C and S2 A). Splenocytes, which are primed throughout life, consistently activated caspases in response to damage. We utilized immunohistochemical (IHC) staining for cleaved caspase-3 and TUNEL to confirm that the cells undergoing apoptosis in each tissue in response to radiation are not blood cells ( Figures 2 D–2F and S2 B–S2F).

Young children frequently experience neuro- and cardiotoxicity when treated with radiation or genotoxic chemotherapies. We therefore hypothesized that the apoptotic pathway may be more active in these tissues in young mammals compared with adults. To test this, we measured levels of apoptotic priming in newborn mice and found that brain, heart, and kidney mitochondria in embryonic and very young mice are extremely primed for apoptosis ( Figure 2 A ). In fact, mitochondria in neonatal brain cells were almost as primed for apoptosis as adult splenocytes. We next sought to delineate the transition between being highly primed and apoptosis refractory in relevant tissues by making serial measurements over time. Each tissue exhibited a distinct temporal program to transition from being primed to being apoptosis refractory, with the sharpest decreases occurring shortly after birth (post-natal day 0–5 [P0–P5]) ( Figure 2 B). In contrast, the spleen maintained a high level of priming throughout development and into adulthood ( Figure 2 B).

(E and F) Cleaved caspase-3-positive (E) or TUNEL-positive (F) cells were counted per 40× field in sham-treated or irradiated tissues from young (P0–P2) or adult (P60+) mice processed via IHC. Data are compiled from two IEs with bars representing means. See Figure S2

(D) After whole-body irradiation, apoptosis was detected via IHC for cleaved caspase-3. Representative images, two IEs. Olfactory bulb (OB) and cerebral cortex (CC) are indicated. Scale bars, 200 μm.

(C) After whole-body irradiation or doxorubicin injection, tissues were collected, and caspase activity was measured via an enzymatic assay. Each point represents an average of three measurements in each mouse tissue across at least five IEs.

(B) Summary BH3 profiling data across tissues during embryonic and post-natal development. Each point represents an average of three measurements in each mouse tissue across at least five IEs.

(A) Tissues were isolated from post-natal day 0 mice (P0) and BH3 profiled. Representative traces are shown (mean ± SD, three or more IEs).

To test whether the lack of BAX and BAK in apoptosis-refractory tissues was preventing activator-induced MOMP, we tested whether addition of recombinant BAX to mitochondria would restore sensitivity to BH3 peptides. While recombinant BAX alone did not induce MOMP, we detected efficient and complete MOMP in adult brain, heart, and kidney tissues when administering BAX concurrently with even minute amounts of BIM ( Figure 1 G). These data indicate that neither BAX nor BAK are expressed at levels sufficient for MOMP in adult brain, heart, and kidney tissues. Moreover, in these tissues there is lower expression of nearly all proteins comprising the mitochondrial apoptotic machinery, both pro- and anti-apoptotic, and pre- and post-mitochondrial. This apoptotic resistance presumably protects vital, long-lived cells constituting these largely post-mitotic tissues from aberrant cell death. Operationally, we defined cells as apoptosis refractory when MOMP is not induced by BIM or BID BH3 at even 100 μM, yet efficiently induced by low doses (1 μM) of BIM BH3 in the presence of exogenous BAX. It is important to note that although cells may be designated as apoptosis refractory based on the BH3 profiling assay, this designation is made at a single time point and in the absence of stress. There may exist conditions under which apoptosis-refractory cells upregulate critical pro-apoptotic proteins to acquire apoptotic sensitivity.

We next examined the levels of these proteins in human tissues by mining mass spectrometry-based proteome data () and found that adult human tissues exhibit a similar pattern of protein expression to that of mice ( Figure 1 F and Tables S1 and S2 ), suggesting that mouse and human tissues regulate apoptosis similarly, which is consistent with previous work ().

We next asked whether the profound differences in mitochondrial priming in adult tissues could be due to differences in expression of BCL-2 family proteins. While we might have expected to find reduced expression of pro-apoptotic proteins or increased expression of their anti-apoptotic counterparts, we instead found that both pro- and anti-apoptotic proteins were lacking in apoptosis-refractory tissues ( Figure 1 E). BAX and BAK, two proteins that are required for mitochondrial apoptosis, were nearly undetectable in refractory tissues. These tissues also expressed lower levels of caspase-3 and -8 to potentially further suppress cell death. We did not observe major differences in the expression of XIAP, a potent inhibitor of caspase activity, across adult tissues.

We previously found that apoptotic priming is a strong determinant of cancer cell fate in response to cytotoxic chemotherapies. We hypothesized that healthy tissue sensitivity to genotoxic agents would be dependent on their degree of priming. To test this hypothesis, we exposed live adult mice to 8 Gy of gamma radiation and found that the degree of apoptosis induced in the tissues corresponded to their level of apoptotic priming ( Figures 1 C, 1D, S1 B, and S1C).

We first performed BH3 profiling on a comprehensive set of adult mouse tissues to detect any potential differences in their apoptotic priming. Cells of the hematopoietic lineage from the periphery (peripheral blood mononuclear cells), thymus, spleen, and bone marrow are the most primed cells in the body among those we studied, as indicated by high mitochondrial depolarization in response to BIM or BID BH3 peptides or full-length proteins ( Figures 1 A , 1B, and S1 A). Cells (excluding blood) constituting the large intestine, small intestine, lungs, and liver were relatively unprimed, as they required higher doses of BIM or BID BH3 peptides and a longer time period to trigger depolarization. Strikingly, we found that adult brain, heart, and kidney tissues are far less primed, and nearly completely insensitive to concentrations of BIM and BID BH3 (100 μM) that are sufficient to induce MOMP in nearly every cancer cell line or primary cancer cell we have tested.

(G) Tissues were isolated from adult mice and BH3 profiled in the presence of recombinant BAX. Representative traces are shown (mean ± SD, three or more IEs). See Figure S1 and Tables S1 and S2

(E) Tissues were collected from adult mice and immunoblotting was performed. Densitometry was performed across immunoblots from three IEs and averaged (blue heatmap on right). GAPDH is loading control. Relative molecular weight markers are shown on left.

(D) After whole-body irradiation, apoptosis was detected via immunohistochemistry for cleaved caspase-3. Data were compiled from two animals for each treatment across two IEs with bars representing means.

(C) After whole-body gamma irradiation, apoptosis was measured via a caspase-3 activity assay. Data were compiled from five animals for each treatment across three IEs with bars representing means. Significance: ∗∗∗∗ p < 0.0001.

(A) Tissues were isolated from adult mice (>P60) and BH3 profiled. Mitochondrial potential under each treatment condition was measured every 5 min during the experimental time course and plotted relative to maximum value of negative control (DMSO). Percent depolarization (indicative of MOMP) is calculated for each BH3 peptide treatment relative to DMSO (0%) and FCCP (100%). Representative traces are shown (mean ± SD, three or more independent experiments [IEs]).

Discussion

Daley, 2009 Daley G.Q. Common themes of dedifferentiation in somatic cell reprogramming and Cancer common themes of dedifferentiation in somatic cell reprogramming and cancer. The study of apoptosis has been traditionally dominated by the study of cancer cells and lymphocytes, the ubiquitous presence of functioning machinery of the mitochondrial apoptotic pathway in which has led to a general acceptance that this pathway is present in all cells. We were surprised to find that heart, kidney, and brain in adult mice lacked the proteins that regulate the mitochondrial apoptotic pathway. It appears that the mitochondrial apoptotic machinery is largely absent in these adult tissues, which we designate as being “apoptosis refractory.” This apoptosis-refractory state is achieved as part of a regulated post-natal developmental program in both mice and humans and is apparently reversible, as stress can restore apoptotic sensitivity to cells previously apoptosis refractory. A common feature of embryonic, immediately post-natal, and regenerating tissues is a greater degree of proliferation. We believe that in all of these tissues, enhanced priming is the price that cells pay for the capacity to proliferate. Cancer cells often share many features of embryonal cells, including lack of differentiation, enhanced proliferation, and stem-like function (). The relatively primed nature of cancer cells may be thought of as re-establishment of yet another embryonic program in cancer cells.

Very young pediatric cancer patients are subject to severe side effects of radiation and chemotherapy from which older children and adults are relatively spared. We show that the apoptotic responses of heart and brain tissues depend on the developmentally regulated priming of the mitochondria. It is possible that this regulation is a major contributor to the hypersensitivity of very young tissues to genotoxic agents. We find this correlation holds in the brains of very young humans. Moreover, we find we can reverse this phenotype by un-priming mitochondria of young tissues by genetic means, potentially modeling pharmacologic inhibition.

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Opferman J.T. Deletion of MCL-1 causes lethal cardiac failure and mitochondrial dysfunction. While our results show that many healthy tissues are apoptosis refractory in the adult, we do not mean to rule out the possibility that various chronic injuries or disease states might restore apoptotic sensitivity in these tissues. In fact, increased levels of neuronal BAX and/or BAK have been reported in patients with Alzheimer's disease (), which would be expected to contribute to the neuronal death that characterizes this pathology. BAX expression has also been observed to increase in different liver pathologies, including hepatitis and cirrhosis (), and is increased following Mcl1 deletion in mouse cardiomyocytes, potentially contributing to the apoptosis reported in that model (). Indeed, we found that chemical liver injury and the resulting regenerative proliferation increased apoptotic priming in adult mice. Such findings suggest that the program we observe is reversible, as we observed with the increased priming post-activation of Myc in the regenerating liver or MycER mouse model. More study is needed to understand how disease states may impact apoptotic priming.

Our analysis shows that virtually all cells within neonatal brain, heart, and kidney are primed for apoptosis while in adults virtually all cells within these tissues are apoptosis refractory. We did not measure priming levels or changes in priming among different types of cells within each tissue. It remains possible, however, that apoptotic priming of cell subsets are differentially regulated in development. Future work will focus on mapping how individual cell types within organs regulate apoptosis.

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et al. Bax channel inhibitors prevent mitochondrion-mediated apoptosis and protect neurons in a model of global brain ischemia. Lessene, 2015 Lessene G. Targeting cell death pathways with small molecules: playing with life and death at the cellular level to treat diseases. Our findings suggest that there may potentially be ways to improve the therapeutic index of cancer treatments through the use of agents that selectively modulate BCL-2 family member function. For example, because of the developing brain's dependence on BAX and not BAK for apoptosis, clinicians could utilize a BAX inhibitor or RNAi-based knockdown of BAX while administering brain irradiation for the treatment or prophylaxis of acute lymphocytic leukemia (ALL) with CNS involvement. This strategy would potentially prevent apoptosis in neurons, which express only BAX, while still allowing ALL cells, which express both BAX and BAK (), to undergo apoptosis via BAK. It may also be possible to modulate developmental programs or Myc levels via recently developed strategies () to induce transient apoptotic resistance in healthy tissues prior to administering radiation or chemotherapy. Beyond cancer, our data have implications for medical conditions such as head trauma in pediatric patients, which induces neuronal death via apoptotic and excitotoxic pathways, while in adults only the excitotoxic pathway is engaged (). A BAX (or BAX/BAK) inhibitor could prevent the apoptotic component of this neuronal death, potentially rescuing some of the ill effects of these injuries in young children. Although no inhibitors of BAX and/or BAK have reached the clinic, efforts are currently underway to develop such agents ().