Tetraploidy in normal tissues and diseases: mechanisms and consequences

Background: Despite the well-documented benefits of exercise and sports participation, young athletes are particularly vulnerable to musculoskeletal injuries. This is especially true during periods of rapid growth, sports specialisation, and high training loads. While injuries are an inevitable aspect of sports participation, the risk can be minimised by promoting the development of strong, resilient tissues through proper nutrition and injury prevention strategies. Moreover, targeted nutrition strategies can accelerate recovery and rehabilitation, allowing for a quicker return to sports participation. Methods: This narrative review synthesises scientific evidence with practical insights to offer comprehensive dietary recommendations aimed at strengthening tissues and supporting the healing process during recovery and rehabilitation. The selection of all sources cited and synthesised in this narrative review were agreed upon by contributing author consensus, experts in sports nutrition (R.A., H.V., B.D.) and exercise and sports medicine (M.H.). Results: Key topics include factors that contribute to injury susceptibility, general dietary recommendations for growth and development, sports nutrition guidelines, and nutrition considerations during injury and rehabilitation. This review also addresses external factors that may lead to suboptimal nutrition, such as food literacy and eating disorders. Conclusions: By highlighting these factors, this article aims to equip coaches, nutritionists, dietitians, athletic trainers, physical therapists, parents/guardians, sporting organisations, and schools with essential knowledge to implement effective nutritional strategies for injury prevention, recovery, and rehabilitation, ultimately enhancing long-term health and athletic performance.

C-kit+ cardiac stem cells, herein called cardiac progenitor cells (CPCs), are beneficial when administered to infarcted mouse hearts. Though the mechanism of these benefits is unknown, CPC vitality likely plays a major role. Thus, investigating the factors governing CPC survival in the ischemic heart may lead to more effective therapeutic strategies. Our previous studies showed that misfolded proteins accumulate in the sarco/endoplasmic reticulum (SR/ER) of the ischemic heart, and that SR/ER protein quality control, which includes the adaptive ER stress response, is critical for the synthesis of most secreted proteins, receptors, and contractile calcium-handling proteins. The transcription factor, ATF6, is a key component of the adaptive ER stress response because it induces genes that reduce the accumulation of misfolded proteins, improving myocyte survival during ischemic stress. While our lab has shown that, in cardiac myocytes, ATF6 is cardioprotective in the ischemic heart, neither the ER stress response nor ATF6 have been examined in CPCs. Accordingly, the hypothesis of this study is that ATF6 and the adaptive ER stress response are critical for optimal survival of CPCs. To address this hypothesis we compared the viabilities of mouse CPCs to neonatal rat ventricular myocytes (NRVM) subjected to treatments that mimic ischemic ER stress in the heart. We found that, compared to NRVM, CPCs exhibited lower levels of adaptive ER stress response gene expression and increased cell death in response to ER stress. Thus, relative to NRVM, the adaptive ER stress response is not fully developed in CPCs. We also found that either siRNA-mediated knock down of ATF6 or chemical inhibition of ATF6 activation with AEBSF decreased adaptive ER stress response gene expression; therefore, ATF6 is an essential component of the adaptive ER stress response in CPCs. Strikingly, ATF6 knockdown decreased CPC viability and proliferation by as much as 70%. Thus, compared to cardiac myocytes, CPCs exhibit a reduced adaptive ER stress response and are more sensitive to ER stress, suggesting that enhancement of the ATF6-mediated adaptive ER stress response in CPCs may be a viable therapeutic approach for enhancing stem cell-mediated myocardial repair.

Entering pregnancy with overweight, obesity or gaining excessive gestational weight could increase the risk of gestational diabetes mellitus (GDM), which is associated with negative consequences for both the mother and the offspring. The objective of this article was to review scientific evidence regarding the association between obesity and GDM, and how weight management through nutritional prevention strategies could prove successful in reducing the risk for GDM. Studies published between January 1975 and January 2009 on the relationship between GDM, pre-pregnancy body mass index (BMI), gestational weight gain and nutritional prevention strategies were included in this review. Results from these reports suggest that maternal obesity assessed by pre-pregnancy BMI is associated with an increased risk of GDM. They also show an association between gestational weight gain and increased risk for GDM. Higher dietary fat and lower carbohydrate intakes during pregnancy appear to be associated with a higher risk for GDM, independent of pre-pregnancy BMI. Some studies showed that restricting energy and carbohydrates could minimize gestational weight gain. However, a firm conclusion on the most effective nutritional intervention for the control of gestational weight gain and glycaemic responses could not be reached based on available studies. In light of the studies reviewed, we conclude that weight management through nutritional prevention strategies could be successful in reducing the risk of GDM. Further studies are required to identify the most effective diet composition to prevent GDM and excessive gestational weight gain.

Rationale: Cardiac stem cells (CSCs) are beneficial when administered to infarcted mouse or rat hearts. Though the mechanism of these benefits is unknown, CSC vitality likely plays a major role. Thus, investigating the factors governing CSC survival in the ischemic heart may lead to more effective therapeutic strategies. Our previous studies showed that misfolded proteins accumulate in the sarco/endoplasmic reticulum (SR/ER) of the ischemic heart. The transcription factor, ATF6, is a key component of the adaptive ER stress response because it induces genes that reduce the accumulation of misfolded proteins, improving myocyte survival during ischemic stress. While our lab has shown that, in cardiac myocytes, ATF6 is cardioprotective in the ischemic heart, neither the ER stress response nor ATF6 have been examined in CSCs. We hypothesize that ATF6 and the adaptive ER stress response are critical for optimal survival of CSCs. Objective/Methods: To gauge the relevance of the ER stress response in CSCs, we used MTT assays to compare the viabilities of mouse CSCs to neonatal rat ventricular myocytes (NRVM) subjected to treatments that mimic ischemic ER stress in the heart. We also assessed the effect of inhibiting ATF6 on both the ER stress response and CSC viability by using chemical inhibition of ATF6 activation or siRNA-mediated ATF6 knock down. Results: We found that, compared to NRVM, CSCs exhibited lower levels of adaptive ER stress response gene expression and decreased viability in response to ER stress. Thus, relative to NRVM, the adaptive ER stress response is not fully developed in CSCs. We also found that either chemical inhibition of ATF6 activation or ATF6 knock down decreased adaptive ER stress response gene expression. Strikingly, ATF6 inhibition or knockdown decreased CSC viability and cell number by as much as 70%. Conclusions: Thus, compared to cardiac myocytes, CSCs exhibit a reduced adaptive ER stress response and are more sensitive to ER stress, suggesting that enhancement of the ATF6-mediated adaptive ER stress response in CSCs may be a viable therapeutic approach for enhancing stem cell-mediated myocardial repair.

The food-borne pathogen Bacillus cereus can acquire enhanced thermal resistance through multiple mechanisms. Two Bacillus cereus strains, ATCC 10987 and ATCC 14579, were used to quantify the effects of salt stress and physiological state on thermotolerance. Cultures were exposed to increasing concentrations of sodium chloride for 30 min, after which their thermotolerance was assessed at 50 degrees C. Linear and nonlinear microbial survival models, which cover a wide range of known inactivation curvatures for vegetative cells, were fitted to the inactivation data and evaluated. Based on statistical indices and model characteristics, biphasic models with a shoulder were selected and used for quantification. Each model parameter reflected a survival characteristic, and both models were flexible, allowing a reduction of parameters when certain phenomena were not present. Both strains showed enhanced thermotolerance after preexposure to (non)lethal salt stress conditions in the exponential phase. The maximum adaptive stress response due to salt preexposure demonstrated for exponential-phase cells was comparable to the effect of physiological state on thermotolerance in both strains. However, the adaptive salt stress response was less pronounced for transition- and stationary-phase cells. The distinct tailing of strain ATCC 10987 was attributed to the presence of a subpopulation of spores. The existence of a stable heat-resistant subpopulation of vegetative cells could not be demonstrated for either of the strains. Quantification of the adaptive stress response might be instrumental in understanding adaptation mechanisms and will allow the food industry to develop more accurate and reliable stress-integrated predictive modeling to optimize minimal processing conditions.

Inbreeding depression is often intensified under environmental stress (i.e., inbreeding-stress interaction). Although the fitness consequences of this phenomenon are well-described, underlying mechanisms such as an increased expression of deleterious alleles under stress, or a lower capacity for adaptive responses to stress with inbreeding, have rarely been investigated. We investigated a fitness component (egg-to-adult viability) and gene-expression patterns using RNA-seq analyses in noninbred control lines and in inbred lines of Drosophila melanogaster exposed to benign temperature or heat stress. We find little support for an increase in the cumulative expression of deleterious alleles under stress. Instead, inbred individuals had a reduced ability to induce an adaptive gene regulatory stress response compared to controls. The decrease in egg-to-adult viability due to stress was most pronounced in the lines with the largest deviation in the adaptive stress response (R2 = 0.48). Thus, we find strong evidence for a lower capacity of inbred individuals to respond by gene regulation to stress and that this is the main driver of inbreeding-stress interactions. In comparison, the altered gene expression due to inbreeding at benign temperature showed no correlation with fitness and was pronounced in genomic regions experiencing the highest increase in homozygosity.

The ATF4 transcription factor is a key regulator of the adaptive integrated stress response (ISR) induced by various stresses and pathologies. Identification of novel transcription targets of ATF4 during ISR would contribute to the understanding of adaptive networks and help to identify novel therapeutic targets. We were previously searching for genes that display an inverse regulation mode by the transcription factors ATF4 and p53 in response to mitochondrial respiration chain complex III inhibition. Among the selected candidates the human genes for cytokeratine 16 (KRT16), anti-apoptotic protein Niban (FAM129A) and hexokinase HKDC1 have been found highly responsive to ATF4 overexpression. Here we explored potential roles of the induction of KRT16, FAM129A and HKDC1 genes in ISR. As verified by RT-qPCR, a dysfunction of mitochondrial respiration chain and ER stress resulted in a partially ATF4-dependent stimulation of KRT16, FAM129A and HKDC1 expression in the HCT116 colon carcinoma cell line. ISRIB, a specific inhibitor of ISR, was able to downregulate the ER stress-induced levels of KRT16, FAM129A and HKDC1 transcripts. An inhibition of ATF4 by RNAi attenuated the induction of KRT16, FAM129A and HKDC1 mRNAs in response to ER stress or to a dysfunctional mitochondrial respiration. The similar induction of the three genes was observed in another tumor-derived cervical carcinoma cell line HeLa. However, in HaCaT and HEK293T cells that display transformed phenotypes, but do not originate from patient-derived tumors, the ER stress-inducing treatments resulted in an upregulation of FAM129A and HKDC1, but not KRT16 transcripts, By a luciferase reporter approach we identified a highly active ATF4-responsive element within the upstream region of the KRT16 gene. The results suggest a conditional regulation of KRT16 gene by ATF4 that may be inhibited in normal cells, but engaged during cancer progression. Potential roles of KRT16, FAM129A and HKDC1 genes upregulation in adaptive stress responses and pathologies are discussed.

Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4-dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca(2+) levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP-treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin--PI3K - MAPK pathways, and up-regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up-regulated in response to DNP. CREB (cAMP-response element-binding protein) signaling, Arc and brain-derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up-regulated in response to DNP, and DNP-treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP-induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up-regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity. Physiological bioenergetic challenges such as exercise and fasting can enhance neuroplasticity and protect neurons against injury and neurodegeneration. Here, we show that the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) elicits adaptive signaling responses in the cerebral cortex involving activation of Ca(2+) -CREB and autophagy pathways, and inhibition of mTOR and insulin signaling pathways. The molecular reprogramming induced by DNP, which is similar to that of exercise and fasting, is associated with improved learning and memory, suggesting potential therapeutic applications for DNP.

Adaptive cellular stress responses are paramount in the healthy control of cell and tissue homeostasis and generally activated during toxicity in a chemical-specific manner. Here, we established a platform containing a panel of distinct adaptive stress response reporter cell lines based on BAC-transgenomics GFP tagging in HepG2 cells. Our current panel of eleven BAC-GFP HepG2 reporters together contains (1) upstream sensors, (2) downstream transcription factors and (3) their respective target genes, representing the oxidative stress response pathway (Keap1/Nrf2/Srxn1), the unfolded protein response in the endoplasmic reticulum (Xbp1/Atf4/BiP/Chop) and the DNA damage response (53bp1/p53/p21). Using automated confocal imaging and quantitative single-cell image analysis, we established that all reporters allowed the time-resolved, sensitive and mode-of-action-specific activation of the individual BAC-GFP reporter cell lines as defined by a panel of pathway-specific training compounds. Implementing the temporal pathway activity information increased the discrimination of training compounds. For a set of >30 hepatotoxicants, the induction of Srxn1, BiP, Chop and p21 BAC-GFP reporters correlated strongly with the transcriptional responses observed in cryopreserved primary human hepatocytes. Together, our data indicate that a phenotypic adaptive stress response profiling platform will allow a high throughput and time-resolved classification of chemical-induced stress responses, thus assisting in the future mechanism-based safety assessment of chemicals.

The YAP1 and YAP2 genes encode yeast transcription factors of the c-jun family. We show that yeast mutants deleted for either the YAP1 or the YAP2 genes are hypersensitive to oxidants, particularly H2O2, and that these genes play a role in regulating the induction of the H2O2 adaptive stress response in Saccharomyces cerevisiae. They do not significantly affect the regulation of the superoxide adaptive stress response. The intrinsic resistance of stationary-phase and respiring yeast cells towards superoxide anions is unaffected by deletion of the YAP1 and YAP2 genes. However, resistance towards H2O2 under these conditions is significantly reduced. We show that expression of the yeast GSH1 gene (encoding gamma-glutamylcysteine synthetase) and the SSA1 gene (encoding an HSP70 isoform) are induced by oxidants. Unlike the SSA1 and thioredoxin (TRX2) genes, expression of the GSH1 gene is more strongly induced by superoxide anions than by H2O2. In the absence of added oxidants, transcription of the GSH1 gene is reduced in strains carrying the yap1 deletion. However, we show that Yap1 is not required for the superoxide anion-mediated induction of GSH1 gene expression. Furthermore, while the H2O2-mediated induction of SSA1 expression is shown to by YAP1 dependent, the heat-shock-mediated induction of the SSA1 gene does not require YAP1. We also present evidence to show that the YAP2 gene does not regulate the expression of the TRX2, SSA1 or GSH1 genes.

From adaption to death: endoplasmic reticulum stress as a novel target of selective neurodegeneration?

Purpose: Ionizing Radiation (IR)-induced bystander effects and genomic instability have important implication in radiotherapy and radioprotection. Their persistence in the progeny of normal cells may contribute to risk of long-term radiation-related health effect in human, including cancer. Hence, this study investigates the role of gap junction intercellular communication (GJIC) and the quality of radiation in the propagation of stressful effects in the unirradiated bystander cells and their progeny. Material and methods: Human skin fibroblasts in the confluent state were exposed to microbeam irradiations with different linear energy transfer (LET) at mean absorbed dose of 0.4 Gy, in the presence or absence of GJIC inhibitor (AGA) by which 0.036-0.4% of cells were directly targeted by IR. After 4 h irradiation or following 20 population doublings, the cells were harvested and assayed for micronucleus (MN) formation, gene mutation and protein oxidation. Results: Our results showed that high-LET carbon microbeams (LET ∼103 keV/μm) and high-LET proton microbeams (LET ∼11 keV/μm) were more effective than low-LET X ray microbeam (LET ∼6 keV/μm) in the induction of DNA damage (MN formation) in bystander cells. Interestingly, significant attenuation of MN formation occurred in bystander cells in the presence of AGA after proton and carbon microbeams. In contrast, incubation of the cells with AGA did not significantly affect the induction of MN formation in bystander cells during confluent holding after X irradiation. Further, the progeny of bystander cells exposed to X rays or protons showed persistent oxidative stress which correlated MN formation and mutation frequency. Such effects were not observed after irradiation by carbon ions. Importantly, the progeny of bystander cells from cultures exposed to protons or carbon ions under conditions where GJIC was inhibited harbored reduced oxidative and genetic damage. This mitigating effect was not detected when the cultured were exposed to X rays. Taken together, the overall results show the expression of stressful effects in the bystander cells and their progeny are dependent on the radiation quality or LET. Conclusions: Our findings suggest that the involvement of GJIC-dependent of radiation quality in the propagation of radiation induces stressful effects to bystander cells and their progeny. In addition, this work provides a strong support to the fact that carbon ions can significantly reduce the risk of cancer and have potential implications in the therapeutic outcome of radiotherapy compared to X rays or protons. Citation Format: Narongchai Autsavapromporn, Ianik Plante, Cuihua Liu, Teruaki Konishi, Noriko Usami, Tomoo Funayama, Yukio Uchihori, Tom K. Hei, Edouard I. Azzam, Sirikan Yamada, Takeshi Murakami, Masao Suzuki. Bystander effect and genomic instability in human cells and their progeny after irradiation with X rays, protons or carbon ions: role of gap junction communication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2015-1815

Background & Hypothesis: The death rate is higher in Black (B) patients (40%), and, in particular, is two-fold higher among young B women (<50 years) compared to White (W) women with breast cancer. Aggressive breast cancer variants, like hormone receptor-negative and inflammatory breast cancer (IBC), contribute to a race-related survival gap and poor clinical outcomes in African American (AA) patients compared to Whites (W) in the United States. We previously identified the dominant role of XIAP in a tumor cell adaptive stress response (ASR) that promotes nuclear transcription factor (NFκB)-mediated proliferative, invasive, and immunosuppressive signaling in breast cancer progression. Our current work sought to address the urgent need to interrogate molecular mechanisms driving the disparity in both AA and IBC tumor biology by investigating gene expression differences between AA and White tumors and Triple-negative breast cancer (TNBC) and non-TNBC tumors, thereby identifying common genes which may be modifiable risk factors and targets that can be modulated for therapeutic benefits. Methods: We used The Cancer Genome Atlas (TCGA) breast cancer data to conduct a comparative analysis of the expression of 226 ASR genes in breast cancer molecular subtypes, normal-adjacent tissues, and in AA and W patients. The ASR gene list includes 101 XIAP-related, 11 NFκB targets, 10 MNK targets, 33 Oxidative Stress Response (OSR)-related, 13 TGFβ-related, 6 JAG1-Notch targets, and 52 immune-related, in addition to 14 genes that belong to more than one ASR set. In the present study, we focused on primary tumor samples [1090 Primary Solid Tumor] and normal tissue samples adjacent to the tumors [113 Solid Tissue Normal]. Within these, our sample set included 559 lumA, 207 lumB, 82 Her2, 190 Basal, and 40 Normal-like. Racial designations in TCGA are based on patient self-identification. In the present study, the race-related analysis focused on AA and W breast cancer patient datasets from TCGA (179 AA and 744 W). It is important to note that among the 113 Normal-adjacent samples, there are 105 W and only 6 AA samples. Results: Analysis of the ASR gene score in different Breast cancer subtypes revealed that ASR genes associated with oxidative stress and immune response pathways score highly in the basal breast cancer subtype. We also observed differential scores between samples of AA or W. The differential gene expression analysis identified 46-88 genes in the ASR set to be differentially expressed (≥2 fold-change and adjusted p-value <0.05) in breast cancer subtypes when comparing each subtype to normal-adjacent tissue or comparing the subtypes to each other. Pathway analysis of the enriched genes revealed the involvement of the cell cycle, the DNA damage response, the signal transduction, and the regulation of cell death-related processes. Moreover, on average, 20% of the ASR genes showed race-related differential expression. Furthermore, a number of ASR that were differentially expressed in breast cancer subtypes and significantly associated (hazard ratio >1.5 and p-value <0.05) with AA and/or W patient survival. For example, CDC45, MCM3, and CDKN2D were uniquely associated with poor survival in AA, but not in W. Conclusion: Within invasive breast cancer, factors that regulate tumor cell ASR reveal subtype and race-specific differences in expression. Understanding their function and downstream signaling will elucidate the intrinsic mechanisms that drive racial disparities in breast cancer outcomes and have the potential to reveal race-related biomarkers and therapeutic targets. Support: NCI-P20 NCCU-Duke Cancer Disparities Translational Research Partnership (SP; GRD, KPW, JF), DoD-W81XWH-17-1-0297 (GRD), Duke Bridge Funds (GRD). Citation Format: Muthana Al Abo, Larisa Gearhart-Serna, Steven Van Laere, Jennifer Freedman, Steven Patierno, Eun-Sil Hwang, Savitri Krishnamurthy, Kevin Williams, Gayathri Devi. Adaptive stress response genes associated with breast cancer subtypes and survival outcomes reveal race-related differences [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-18.

The illicit generation of tetraploid cells constitutes a prominent driver of oncogenesis, as it often precedes the development of aneuploidy and genomic instability. In addition, tetraploid (pre-)malignant cells display an elevated resistance against radio- and chemotherapy. Here, we report a strategy to preferentially kill tetraploid tumor cells based on the broad-spectrum kinase inhibitor SP600125. Live videomicroscopy revealed that SP600125 affects the execution of mitosis, impedes proper cell division and/or activates apoptosis in near-to-tetraploid, though less so in parental, cancer cells. We propose a novel graphical model to quantify the differential response of diploid and tetraploid cells to mitotic perturbators, including SP600125, which we baptized “transgenerational cell fate profiling.” We speculate that this representation constitutes a valid alternative to classical “single-cell fate” and “genealogical” profiling and, hence, may facilitate the analysis of cell fate within a heterogeneous population as well as the visual examination of cell cycle alterations.

Aneuploidy and chromosomal instability, which are frequent in cancer, can result from the asymmetric division of tetraploid precursors. Genomic instability may favor the generation of more aggressive tumor cells with a reduced propensity for undergoing apoptosis. To assess the impact of tetraploidization on apoptosis regulation, we generated a series of stable tetraploid HCT116 and RKO colon carcinoma cell lines. When comparing diploid parental cells with tetraploid clones, we found that such cells were equally sensitive to a series of cytotoxic agents (staurosporine [STS], hydroxyurea, etoposide), as well as to the lysis by natural killer cells. In strict contrast, tetraploid cells were found to be relatively resistant against a series of DNA-damaging agents, namely cisplatin, oxaliplatin, camptothecin, and gamma- and UVC-irradiation. This increased resistance correlated with a reduced manifestation of apoptotic parameters (such as the dissipation of the mitochondrial transmembrane potential and the degradation of nuclear DNA) in tetraploid as compared to diploid cells subjected to DNA damage. Moreover, tetraploid cells manifested an enhanced baseline level of p53 activation. Inhibition of p53 abolished the difference in the susceptibility of diploid and tetraploid cancer cells to DNA damage-induced apoptosis. These data point to an intrinsic resistance of tetraploid cells against radiotherapy and DNA-targeted chemotherapy that may be linked to the status of the p53 system.