Cold pressor stress effects on cardiac repolarization.

The cold pressor test (CPT) elicits strong cardiovascular reactions via activation of the sympathetic nervous system (SNS), yielding subsequent increases in heart rate (HR) and blood pressure (BP). However, little is known on how exposure to the CPT affects cardiac ventricular repolarization. Twenty-eight healthy males underwent both a bilateral feet CPT and a warm water (WW) control condition on two separate days, one week apart. During pre-stress baseline and stress induction cardiovascular signals (ECG lead II, Finometer BP) were monitored continuously. Salivary cortisol and subjective stress ratings were assessed intermittently. Corrected QT (QTc) interval length and T-wave amplitude (TWA) were assessed for each heartbeat and subsequently aggregated individually over baseline and stress phases, respectively. CPT increases QTc interval length and elevates the TWA. Stress-induced changes in cardiac repolarization are only in part and weakly correlated with cardiovascular and cortisol stress-reactivity. Besides its already well-established effects on cardiovascular, endocrine, and subjective responses, CPT also impacts on cardiac repolarization by elongation of QTc interval length and elevation of TWA. CPT effects on cardiac repolarization share little variance with the other indices of stress reactivity, suggesting a potentially incremental value of this parameter for understanding psychobiological adaptation to acute CPT stress.

Test-retest reproducibility of a combined physical and cognitive stressor.

When studying the factors which influence stress reactivity in within-subject designs, test-retest reproducibility data is needed to estimate power and sample size. We report such data regarding a new experimental stress protocol, based on simultaneous application of the socially evaluated, bilateral feet Cold Pressor Test (CPT) and the Paced Auditory Serial Addition Task (PASAT). Cardiovascular, neuroendocrine, and subjective (affective) stress responses of 32 healthy males were measured twice, at an interval of one week. The novel protocol induced substantial stress reactivity in all parameters at both test and retest. Cardiovascular reactivity remained unchanged, but cortisol and subjective responses were lower at second stress exposure, with high test-retest stability of neuroendocrine (r>.7) and cardiovascular measures (r = .5 to r = .9). PASAT performance improved. Response attenuation suggests habituation-like and/or learning effects. Data provided by our study demonstrate feasibility and power of this stress protocol for investigating changes in stress reactivity in repeated, within-subject designs.

Emotional stress regulation: The role of relative frontal alpha asymmetry in shaping the stress response.

Frontal EEG asymmetry has been proposed as an index of emotional regulation, reflecting both state and trait components, and there is evidence that these factors influence the cortisol response to stress. Here, we asked whether cold pressor stress modulates frontal asymmetry and whether this is predictive of the neuroendocrine stress response. Twenty-four male participants underwent an automatized bilateral feet cold pressor test (bfCPT) and a warm water control procedure in counterbalanced order on two separate days, one week apart. EEG, heart rate and blood pressure were assessed at baseline as well as during and after the bfCPT. Salivary cortisol and subjective ratings of stress and arousal were assessed before and after the bfCPT. The bfCPT led to a significant increase in cortisol, cardiovascular parameters and in subjective ratings of stress and arousal that was absent in the control condition. Furthermore, analysis of relative frontal alpha-band asymmetry revealed a stronger relative right frontal activation during the bfCPT compared to the control condition at electrode pairs F7/8 but not F3/4. However, frontal asymmetry scores during the bfCPT were not predictive for neither physiological responses nor subjective ratings. Moreover, an association between physiological responses and frontal asymmetry assessed during rest at baseline could be observed at electrode pairs F3/F4, with stronger responses being associated with stronger relative right frontal activation. Our results show that cold pressor stress leads to an alteration of emotional processes as reflected in frontal EEG asymmetry at F7/F8. Moreover, physiological responses to the CPT seem to be differentially moderated by trait and state components present in frontal asymmetry.

Blunted endocrine response to a combined physical-cognitive stressor in adults with early life adversity.

The negative health effects of early life adversity (ELA) continue long into adulthood. Changes in the physiological response to psychosocial stressors have been proposed to mediate this effect. However, many previous studies have come to contradicting conclusions as to whether ELA induces a long-term increase or decrease in stress reactivity. Therefore, we tested the association of ELA exposure and adult stress reactivity in a sample of early life adoptees and controls. Two previously validated stressful elements (bilateral feet CPT and the Paced Auditory Serial Addition Task (PASAT)) were combined in an extended Cold Pressor Test (CPT). This test was performed on 22 participants who had experienced severe ELA (separation from biological parents, institutionalization, and adoption in early childhood), and in 22 age-matched control participants. A prior history of ELA was associated with blunted reactivity of the hypothalamic-pituitary-adrenal (HPA) axis (Cohen´s d = 0.680). Cardiovascular reactivity remained unchanged, and affective reactivity (self-report ratings) were increased in participants exposed to ELA compared to the control group (range Cohen´s d: 0.642-0.879). Our results suggest that the activity of the HPA axis reactivity was inhibited in ELA participants. Importantly, cardiovascular stress responsiveness was not affected by ELA. This separation of the HPA axis and cardiovascular stress responses may best be explained by ELA selectively enhancing central feedback-sensitivity to glucocorticoids, but preserving cardiovascular/ autonomic stress reactivity.

Validation of an automated bilateral feet cold pressor test.

The Cold Pressor Test (CPT) is often used in psychobiological research. However, the classical CPT version (unilateral hand immersion into ice-water) involves some disadvantages: hands may be needed for further applications, attentional drift towards the affected sensory hemi-field and/or physiological activation of the contralateral hemisphere may produce a laterality bias. Furthermore, instruction-induced motor activity may bias physiologic reactivity. To avoid these problems, a fully automated bilateral feet CPT was developed and tested for validity and feasibility. The test procedure is based on computerized control of water influx and efflux. This allows for maximal standardization and precise timing. Furthermore, water is kept in permanent flow to prohibit formation of stable temperature layers in skin proximity. Laterality bias, instructions effects and motor responses (e.g. lifting feet out of a water basin) are avoided. In a counterbalanced within-subject design, 28 healthy male students were exposed to the CPT and to a warm water control (CNT) condition twice, one week apart. Cardiovascular parameters, salivary cortisol and subjective ratings (stress, arousal and pain) were assessed before, during, and after interventions. The CPT profoundly affected physiology as well as subjective ratings. Expectation effects (immediately before testing) were small. Furthermore, post-CPT (presumably compensatory/counter-regulatory) effects on heart rate and stroke volume were found. In conclusion, the automated bilateral feet CPT is a valid and feasible stress test modification. Hemodynamic, subjective and endocrine stress responses are substantial, suggesting that this test version represents an advanced and suitable tool in human stress research.

Epigenetic silencing of the proapoptotic gene BIM in anaplastic large cell lymphoma through an MeCP2/SIN3a deacetylating complex.

BIM is a proapoptotic member of the Bcl-2 family. Here, we investigated the epigenetic status of the BIM locus in NPM/ALK+ anaplastic large cell lymphoma (ALCL) cell lines and in lymph node biopsies from NPM/ALK+ ALCL patients. We show that BIM is epigenetically silenced in cell lines and lymph node specimens and that treatment with the deacetylase inhibitor trichostatin A restores the histone acetylation, strongly upregulates BIM expression, and induces cell death. BIM silencing occurs through recruitment of MeCP2 and the SIN3a/histone deacetylase 1/2 (HDAC1/2) corepressor complex. This event requires BIM CpG methylation/demethylation with 5-azacytidine that leads to detachment of the MeCP2 corepressor complex and reacetylation of the histone tails. Treatment with the ALK inhibitor PF2341066 or with an inducible shRNA targeting NPM/ALK does not restore BIM locus reacetylation; however, enforced expression of NPM/ALK in an NPM/ALK-negative cell line significantly increases the methylation at the BIM locus. This study demonstrates that BIM is epigenetically silenced in NPM/ALK-positive cells through recruitment of the SIN3a/HDAC1/2 corepressor complex and that NPM/ALK is dispensable to maintain BIM epigenetic silencing but is able to act as an inducer of BIM methylation.

p53-independent epigenetic repression of the p21(WAF1) gene in T-cell acute lymphoblastic leukemia.

The p53 protein is a primary mediator of cellular apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has shown that the majority of childhood acute lymphoblastic leukemia (ALL) cases express a wild type p53 gene, although the functionality of the p53 pathway has rarely been validated. In the present study, the integrity of the p53 pathway was investigated in a panel of ALL cell lines and xenografts established from direct patient explants in immune-deficient mice. A focused real-time quantitative reverse transcription PCR array of known p53-regulated genes identified p21(WAF1) (CDKN1A) as the highest ranked gene to be differentially expressed between B-cell precursor (BCP)-ALL and T-ALL xenografts following exposure to the DNA-damaging drug etoposide. Lack of p21(WAF1) induction was observed in six of seven T-ALL xenograft lines, as well as primary T-ALL cells following irradiation exposure, despite an otherwise functional p53 response. Repression of p21(WAF1) in T-ALL cells was associated with decreased acetylated H3K9 localized at its promoter compared with BCP-ALL cells, together with increased CpG methylation within the first exon and intron. Although the histone deacetylase inhibitor vorinostat failed to induce p21(WAF1) in T-ALL samples, the combination of vorinostat and the demethylating agent decitabine reactivated expression of the silenced p21(WAF1) gene in the Molt-4 T-ALL cell line. Considering the known anti-apoptotic function of p21(WAF1), our findings have significant implications for the responses of T- versus BCP-ALL cells to chemotherapeutic drugs that induce p21(WAF1).

Epigenetic silencing of BIM in glucocorticoid poor-responsive pediatric acute lymphoblastic leukemia, and its reversal by histone deacetylase inhibition.

Glucocorticoids play a critical role in the therapy of lymphoid malignancies, including pediatric acute lymphoblastic leukemia (ALL), although the mechanisms underlying cellular resistance remain unclear. We report glucocorticoid resistance attributable to epigenetic silencing of the BIM gene in pediatric ALL biopsies and xenografts established in immune-deficient mice from direct patient explants as well as a therapeutic approach to reverse resistance in vivo. Glucocorticoid resistance in ALL xenografts was consistently associated with failure to up-regulate BIM expression after dexamethasone exposure despite confirmation of a functional glucocorticoid receptor. Although a comprehensive assessment of BIM CpG island methylation revealed no consistent changes, glucocorticoid resistance in xenografts and patient biopsies significantly correlated with decreased histone H3 acetylation. Moreover, the histone deacetylase inhibitor vorinostat relieved BIM repression and exerted synergistic antileukemic efficacy with dexamethasone in vitro and in vivo. These findings provide a novel therapeutic strategy to reverse glucocorticoid resistance and improve outcome for high-risk pediatric ALL.

In vivo models of childhood leukemia for preclinical drug testing.

The number of new anti-cancer drugs emerging for clinical trials in humans far exceeds the availability of pediatric acute leukemia patients to be entered into clinical trials. Therefore, preclinical testing of new agents for the treatment of childhood acute leukemia is essential to ensure that the most promising drugs are prioritized to enter clinical trials. Historically, the murine system has been central to modeling human leukemia in vivo. A greater knowledge of the molecular lesions underlying particular subtypes of leukemia has led to the generation of genetically engineered murine models, generally involving the knockin or knockout of certain genes and fusion genes at their normal genetic locus. However, the most predominant in vivo models for preclinical drug testing have been human leukemia xenografts. Successful engraftment of all subtypes of acute lymphoblastic leukemia, most subtypes of acute myeloid leukemia as well as juvenile myelomonocytic leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia have been described in various immune-deficient murine hosts. Preclinical testing of novel therapeutics in vivo will likely identify the most promising new agents to enter clinical trials, and will allow their future use to be optimized in combination with other novel and conventional chemotherapeutics.

Divergent mechanisms of glucocorticoid resistance in experimental models of pediatric acute lymphoblastic leukemia.

Cell line models of glucocorticoid resistance in childhood acute lymphoblastic leukemia (ALL) almost invariably exhibit altered glucocorticoid receptor (GR) function. However, these findings are incongruous with those using specimens derived directly from leukemia patients, in which GR alterations are rarely found. Consequently, mechanisms of glucocorticoid resistance in the clinical setting remain largely unresolved. We present a novel paradigm of glucocorticoid resistance in childhood ALL, in which patient biopsies have been directly established as continuous xenografts in immune-deficient mice, without prior in vitro culture. We show that the GRs from six highly dexamethasone-resistant xenografts (in vitro IC(50) >10 micromol/L) exhibit no defects in ligand-induced nuclear translocation and binding to a consensus glucocorticoid response element (GRE). This finding contrasts with five commonly used leukemia cell lines, all of which exhibited defective GRE binding. Moreover, whereas the GRs of dexamethasone-resistant xenografts were transcriptionally active, as assessed by the ability to induce the glucocorticoid-induced leucine zipper (GILZ) gene, resistance was associated with failure to induce the bim gene, which encodes a proapoptotic BH3-only protein. Furthermore, the receptor tyrosine kinase inhibitor, SU11657, completely reversed dexamethasone resistance in a xenograft expressing functional GR, indicating that pharmacologic reversal of glucocorticoid resistance in childhood ALL is achievable.

Xenograft models for the preclinical evaluation of new therapies in acute leukemia.

Major advances in understanding the pathophysiology of acute leukemia have resulted in a dramatic increase in the availability of novel compounds for clinical trials. However, since the number of new drugs far exceeds the number of clinical trials that can be conducted because of the availability of eligible patients, there is an urgent need to utilize reliable preclinical models for the prioritization of the most promising potential therapies for those clinical trials. The most widely used preclinical models for the acute leukemias are human tumor xenografts established in immune-deficient mice, and genetically engineered mouse strains. This review summarizes the recent developments and considerations in the use of xenograft models of acute lymphoblastic leukemia, acute myeloid leukemia, and acute promyelocytic leukemia for the preclinical testing of new therapies.

Dexamethasone resistance in B-cell precursor childhood acute lymphoblastic leukemia occurs downstream of ligand-induced nuclear translocation of the glucocorticoid receptor.

Glucocorticoids are among the most effective agents used in the treatment of childhood acute lymphoblastic leukemia (ALL), and patient response to treatment is an important determinant of long-term outcome. Despite its clinical significance, the molecular basis of glucocorticoid resistance in lymphoid malignancies is still poorly understood. We have recently developed a highly clinically relevant experimental model of childhood ALL, in which primary childhood ALL biopsies were established as xenografts in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The in vivo and in vitro responses of a panel of these xenografts to the glucocorticoid, dexamethasone, reflected the outcome of the patients from whom they were derived. In this report we show that glucocorticoid resistance in B-cell precursor (BCP) ALL xenografts was not due to down-regulation of the glucocorticoid receptor (GR) nor to defective ligand binding of the GR. Moreover, dexamethasone-induced GR translocation from the cytoplasm to the nucleus was comparable in all xenografts. However, glucocorticoid resistance was associated with profoundly attenuated induction of the BH3-only proapoptotic protein, Bim, when xenograft cells were exposed to dexamethasone. These results show that dexamethasone resistance in BCP ALL xenografts occurs downstream of ligand-induced nuclear translocation of the GR, but upstream of Bim induction.