Temporal effects on death by suicide: empirical evidence and possible molecular correlates.

Popular culture and medical lore have long postulated a connection between full moon and exacerbations of psychiatric disorders. We wanted to empirically analyze the hypothesis that suicides are increased during the period around full moons. We analyzed pre-COVID suicides from the Marion County Coroner's Office (n = 776), and show that deaths by suicide are significantly increased during the week of the full moon (p = 0.037), with older individuals (age ≥ 55) showing a stronger effect (p = 0.019). We also examined in our dataset which hour of the day (3-4 pm, p = 0.035), and which month of the year (September, p = 0.09) show the most deaths by suicide. We had blood samples on a subset of the subjects (n = 45), which enabled us to look at possible molecular mechanisms. We tested a list of top blood biomarkers for suicidality (n = 154) from previous studies of ours 7, to assess which of them are predictive. The biomarkers for suicidality that are predictive of death by suicide during full moon, peak hour of day, and peak month of year, respectively, compared to outside of those periods, appear to be enriched in circadian clock genes. For full moon it is AHCYL2, ACSM3, AK2, and RBM3. For peak hour it is GSK3B, AK2, and PRKCB. For peak month it is TBL1XR1 and PRKCI. Half of these genes are modulated in expression by lithium and by valproate in opposite direction to suicidality, and all of them are modulated by depression and alcohol in the same direction as suicidality. These data suggest that there are temporal effects on suicidality, possibly mediated by biological clocks, pointing to changes in ambient light (timing and intensity) as a therapeutically addressable target to decrease suicidality, that can be coupled with psychiatric pharmacological and addiction treatment preventive interventions.

Precision medicine for suicidality: from universality to subtypes and personalization.

Suicide remains a clear, present and increasing public health problem, despite being a potentially preventable tragedy. Its incidence is particularly high in people with overt or un(der)diagnosed psychiatric disorders. Objective and precise identification of individuals at risk, ways of monitoring response to treatments and novel preventive therapeutics need to be discovered, employed and widely deployed. We sought to investigate whether blood gene expression biomarkers for suicide (that is, a 'liquid biopsy' approach) can be identified that are more universal in nature, working across psychiatric diagnoses and genders, using larger cohorts than in previous studies. Such markers may reflect and/or be a proxy for the core biology of suicide. We were successful in this endeavor, using a comprehensive stepwise approach, leading to a wealth of findings. Steps 1, 2 and 3 were discovery, prioritization and validation for tracking suicidality, resulting in a Top Dozen list of candidate biomarkers comprising the top biomarkers from each step, as well as a larger list of 148 candidate biomarkers that survived Bonferroni correction in the validation step. Step 4 was testing the Top Dozen list and Bonferroni biomarker list for predictive ability for suicidal ideation (SI) and for future hospitalizations for suicidality in independent cohorts, leading to the identification of completely novel predictive biomarkers (such as CLN5 and AK2), as well as reinforcement of ours and others previous findings in the field (such as SLC4A4 and SKA2). Additionally, we examined whether subtypes of suicidality can be identified based on mental state at the time of high SI and identified four potential subtypes: high anxiety, low mood, combined and non-affective (psychotic). Such subtypes may delineate groups of individuals that are more homogenous in terms of suicidality biology and behavior. We also studied a more personalized approach, by psychiatric diagnosis and gender, with a focus on bipolar males, the highest risk group. Such a personalized approach may be more sensitive to gender differences and to the impact of psychiatric co-morbidities and medications. We compared testing the universal biomarkers in everybody versus testing by subtypes versus personalized by gender and diagnosis, and show that the subtype and personalized approaches permit enhanced precision of predictions for different universal biomarkers. In particular, LHFP appears to be a strong predictor for suicidality in males with depression. We also directly examined whether biomarkers discovered using male bipolars only are better predictors in a male bipolar independent cohort than universal biomarkers and show evidence for a possible advantage of personalization. We identified completely novel biomarkers (such as SPTBN1 and C7orf73), and reinforced previously known biomarkers (such as PTEN and SAT1). For diagnostic ability testing purposes, we also examined as predictors phenotypic measures as apps (for suicide risk (CFI-S, Convergent Functional Information for Suicidality) and for anxiety and mood (SASS, Simplified Affective State Scale)) by themselves, as well as in combination with the top biomarkers (the combination being our a priori primary endpoint), to provide context and enhance precision of predictions. We obtained area under the curves of 90% for SI and 77% for future hospitalizations in independent cohorts. Step 5 was to look for mechanistic understanding, starting with examining evidence for the Top Dozen and Bonferroni biomarkers for involvement in other psychiatric and non-psychiatric disorders, as a mechanism for biological predisposition and vulnerability. The biomarkers we identified also provide a window towards understanding the biology of suicide, implicating biological pathways related to neurogenesis, programmed cell death and insulin signaling from the universal biomarkers, as well as mTOR signaling from the male bipolar biomarkers. In particular, HTR2A increase coupled with ARRB1 and GSK3B decreases in expression in suicidality may provide a synergistic mechanistical corrective target, as do SLC4A4 increase coupled with AHCYL1 and AHCYL2 decrease. Step 6 was to move beyond diagnostics and mechanistical risk assessment, towards providing a foundation for personalized therapeutics. Items scored positive in the CFI-S and subtypes identified by SASS in different individuals provide targets for personalized (psycho)therapy. Some individual biomarkers are targets of existing drugs used to treat mood disorders and suicidality (lithium, clozapine and omega-3 fatty acids), providing a means toward pharmacogenomics stratification of patients and monitoring of response to treatment. Such biomarkers merit evaluation in clinical trials. Bioinformatics drug repurposing analyses with the gene expression biosignatures of the Top Dozen and Bonferroni-validated universal biomarkers identified novel potential therapeutics for suicidality, such as ebselen (a lithium mimetic), piracetam (a nootropic), chlorogenic acid (a polyphenol) and metformin (an antidiabetic and possible longevity promoting drug). Finally, based on the totality of our data and of the evidence in the field to date, a convergent functional evidence score prioritizing biomarkers that have all around evidence (track suicidality, predict it, are reflective of biological predisposition and are potential drug targets) brought to the fore APOE and IL6 from among the universal biomarkers, suggesting an inflammatory/accelerated aging component that may be a targetable common denominator.

Towards understanding and predicting suicidality in women: biomarkers and clinical risk assessment.

Women are under-represented in research on suicidality to date. Although women have a lower rate of suicide completion than men, due in part to the less-violent methods used, they have a higher rate of suicide attempts. Our group has previously identified genomic (blood gene expression biomarkers) and clinical information (apps) predictors for suicidality in men. We now describe pilot studies in women. We used a powerful within-participant discovery approach to identify genes that change in expression between no suicidal ideation (no SI) and high suicidal ideation (high SI) states (n=12 participants out of a cohort of 51 women psychiatric participants followed longitudinally, with diagnoses of bipolar disorder, depression, schizoaffective disorder and schizophrenia). We then used a Convergent Functional Genomics (CFG) approach to prioritize the candidate biomarkers identified in the discovery step by using all the prior evidence in the field. Next, we validated for suicidal behavior the top-ranked biomarkers for SI, in a demographically matched cohort of women suicide completers from the coroner's office (n=6), by assessing which markers were stepwise changed from no SI to high SI to suicide completers. We then tested the 50 biomarkers that survived Bonferroni correction in the validation step, as well as top increased and decreased biomarkers from the discovery and prioritization steps, in a completely independent test cohort of women psychiatric disorder participants for prediction of SI (n=33) and in a future follow-up cohort of psychiatric disorder participants for prediction of psychiatric hospitalizations due to suicidality (n=24). Additionally, we examined how two clinical instruments in the form of apps, Convergent Functional Information for Suicidality (CFI-S) and Simplified Affective State Scale (SASS), previously tested in men, perform in women. The top CFI-S item distinguishing high SI from no SI states was the chronic stress of social isolation. We then showed how the clinical information apps combined with the 50 validated biomarkers into a broad predictor (UP-Suicide), our apriori primary end point, predicts suicidality in women. UP-Suicide had a receiver-operating characteristic (ROC) area under the curve (AUC) of 82% for predicting SI and an AUC of 78% for predicting future hospitalizations for suicidality. Some of the individual components of the UP-Suicide showed even better results. SASS had an AUC of 81% for predicting SI, CFI-S had an AUC of 84% and the combination of the two apps had an AUC of 87%. The top biomarker from our sequential discovery, prioritization and validation steps, BCL2, predicted future hospitalizations due to suicidality with an AUC of 89%, and the panel of 50 validated biomarkers (BioM-50) predicted future hospitalizations due to suicidality with an AUC of 94%. The best overall single blood biomarker for predictions was PIK3C3 with an AUC of 65% for SI and an AUC of 90% for future hospitalizations. Finally, we sought to understand the biology of the biomarkers. BCL2 and GSK3B, the top CFG scoring validated biomarkers, as well as PIK3C3, have anti-apoptotic and neurotrophic effects, are decreased in expression in suicidality and are known targets of the anti-suicidal mood stabilizer drug lithium, which increases their expression and/or activity. Circadian clock genes were overrepresented among the top markers. Notably, PER1, increased in expression in suicidality, had an AUC of 84% for predicting future hospitalizations, and CSNK1A1, decreased in expression, had an AUC of 96% for predicting future hospitalizations. Circadian clock abnormalities are related to mood disorder, and sleep abnormalities have been implicated in suicide. Docosahexaenoic acid signaling was one of the top biological pathways overrepresented in validated biomarkers, which is of interest given the potential therapeutic and prophylactic benefits of omega-3 fatty acids. Some of the top biomarkers from the current work in women showed co-directionality of change in expression with our previous work in men, whereas others had changes in opposite directions, underlying the issue of biological context and differences in suicidality between the two genders. With this study, we begin to shed much needed light in the area of female suicidality, identify useful objective predictors and help understand gender commonalities and differences. During the conduct of the study, one participant committed suicide. In retrospect, when the analyses were completed, her UP-Suicide risk prediction score was at the 100 percentile of all participants tested.

Understanding and predicting suicidality using a combined genomic and clinical risk assessment approach.

Worldwide, one person dies every 40 seconds by suicide, a potentially preventable tragedy. A limiting step in our ability to intervene is the lack of objective, reliable predictors. We have previously provided proof of principle for the use of blood gene expression biomarkers to predict future hospitalizations due to suicidality, in male bipolar disorder participants. We now generalize the discovery, prioritization, validation, and testing of such markers across major psychiatric disorders (bipolar disorder, major depressive disorder, schizoaffective disorder, and schizophrenia) in male participants, to understand commonalities and differences. We used a powerful within-participant discovery approach to identify genes that change in expression between no suicidal ideation and high suicidal ideation states (n=37 participants out of a cohort of 217 psychiatric participants followed longitudinally). We then used a convergent functional genomics (CFG) approach with existing prior evidence in the field to prioritize the candidate biomarkers identified in the discovery step. Next, we validated the top biomarkers from the prioritization step for relevance to suicidal behavior, in a demographically matched cohort of suicide completers from the coroner's office (n=26). The biomarkers for suicidal ideation only are enriched for genes involved in neuronal connectivity and schizophrenia, the biomarkers also validated for suicidal behavior are enriched for genes involved in neuronal activity and mood. The 76 biomarkers that survived Bonferroni correction after validation for suicidal behavior map to biological pathways involved in immune and inflammatory response, mTOR signaling and growth factor regulation. mTOR signaling is necessary for the effects of the rapid-acting antidepressant agent ketamine, providing a novel biological rationale for its possible use in treating acute suicidality. Similarly, MAOB, a target of antidepressant inhibitors, was one of the increased biomarkers for suicidality. We also identified other potential therapeutic targets or biomarkers for drugs known to mitigate suicidality, such as omega-3 fatty acids, lithium and clozapine. Overall, 14% of the top candidate biomarkers also had evidence for involvement in psychological stress response, and 19% for involvement in programmed cell death/cellular suicide (apoptosis). It may be that in the face of adversity (stress), death mechanisms are turned on at a cellular (apoptosis) and organismal level. Finally, we tested the top increased and decreased biomarkers from the discovery for suicidal ideation (CADM1, CLIP4, DTNA, KIF2C), prioritization with CFG for prior evidence (SAT1, SKA2, SLC4A4), and validation for behavior in suicide completers (IL6, MBP, JUN, KLHDC3) steps in a completely independent test cohort of psychiatric participants for prediction of suicidal ideation (n=108), and in a future follow-up cohort of psychiatric participants (n=157) for prediction of psychiatric hospitalizations due to suicidality. The best individual biomarker across psychiatric diagnoses for predicting suicidal ideation was SLC4A4, with a receiver operating characteristic (ROC) area under the curve (AUC) of 72%. For bipolar disorder in particular, SLC4A4 predicted suicidal ideation with an AUC of 93%, and future hospitalizations with an AUC of 70%. SLC4A4 is involved in brain extracellular space pH regulation. Brain pH has been implicated in the pathophysiology of acute panic attacks. We also describe two new clinical information apps, one for affective state (simplified affective state scale, SASS) and one for suicide risk factors (Convergent Functional Information for Suicide, CFI-S), and how well they predict suicidal ideation across psychiatric diagnoses (AUC of 85% for SASS, AUC of 89% for CFI-S). We hypothesized a priori, based on our previous work, that the integration of the top biomarkers and the clinical information into a universal predictive measure (UP-Suicide) would show broad-spectrum predictive ability across psychiatric diagnoses. Indeed, the UP-Suicide was able to predict suicidal ideation across psychiatric diagnoses with an AUC of 92%. For bipolar disorder, it predicted suicidal ideation with an AUC of 98%, and future hospitalizations with an AUC of 94%. Of note, both types of tests we developed (blood biomarkers and clinical information apps) do not require asking the individual assessed if they have thoughts of suicide, as individuals who are truly suicidal often do not share that information with clinicians. We propose that the widespread use of such risk prediction tests as part of routine or targeted healthcare assessments will lead to early disease interception followed by preventive lifestyle modifications and proactive treatment.

Discovery and validation of blood biomarkers for suicidality.

Suicides are a leading cause of death in psychiatric patients, and in society at large. Developing more quantitative and objective ways (biomarkers) for predicting and tracking suicidal states would have immediate practical applications and positive societal implications. We undertook such an endeavor. First, building on our previous blood biomarker work in mood disorders and psychosis, we decided to identify blood gene expression biomarkers for suicidality, looking at differential expression of genes in the blood of subjects with a major mood disorder (bipolar disorder), a high-risk population prone to suicidality. We compared no suicidal ideation (SI) states and high SI states using a powerful intrasubject design, as well as an intersubject case-case design, to generate a list of differentially expressed genes. Second, we used a comprehensive Convergent Functional Genomics (CFG) approach to identify and prioritize from the list of differentially expressed gene biomarkers of relevance to suicidality. CFG integrates multiple independent lines of evidence-genetic and functional genomic data-as a Bayesian strategy for identifying and prioritizing findings, reducing the false-positives and false-negatives inherent in each individual approach. Third, we examined whether expression levels of the blood biomarkers identified by us in the live bipolar subject cohort are actually altered in the blood in an age-matched cohort of suicide completers collected from the coroner's office, and report that 13 out of the 41 top CFG scoring biomarkers (32%) show step-wise significant change from no SI to high SI states, and then to the suicide completers group. Six out of them (15%) remained significant after strict Bonferroni correction for multiple comparisons. Fourth, we show that the blood levels of SAT1 (spermidine/spermine N1-acetyltransferase 1), the top biomarker identified by us, at the time of testing for this study, differentiated future as well as past hospitalizations with suicidality, in a live cohort of bipolar disorder subjects, and exhibited a similar but weaker pattern in a live cohort of psychosis (schizophrenia/schizoaffective disorder) subjects. Three other (phosphatase and tensin homolog (PTEN), myristoylated alanine-rich protein kinase C substrate (MARCKS), and mitogen-activated protein kinase kinase kinase 3 (MAP3K3)) of the six biomarkers that survived Bonferroni correction showed similar but weaker effects. Taken together, the prospective and retrospective hospitalization data suggests SAT1, PTEN, MARCKS and MAP3K3 might be not only state biomarkers but trait biomarkers as well. Fifth, we show how a multi-dimensional approach using SAT1 blood expression levels and two simple visual-analog scales for anxiety and mood enhances predictions of future hospitalizations for suicidality in the bipolar cohort (receiver-operating characteristic curve with area under the curve of 0.813). Of note, this simple approach does not directly ask about SI, which some individuals may deny or choose not to share with clinicians. Lastly, we conducted bioinformatic analyses to identify biological pathways, mechanisms and medication targets. Overall, suicidality may be underlined, at least in part, by biological mechanisms related to stress, inflammation and apoptosis.

The protein tyrosine phosphatase, Shp2, positively contributes to FLT3-ITD-induced hematopoietic progenitor hyperproliferation and malignant disease in vivo.

Internal tandem duplications (ITDs) in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia (AML). We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knockdown of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low-density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML.

Inhibition of glycinamide ribonucleotide formyltransferase results in selective inhibition of macrophage cytokine secretion in vitro and in vivo efficacy in rat adjuvant arthritis.

OBJECTIVE: To determine the effects of a glycinamide ribonucleotide formyltransferase (GARFT) inhibitor on macrophage inflammatory processes and in vivo in rat adjuvant arthritis. METHODS: GARFT inhibitors, LY309886 (6S-2',5'-thienyl-5, 10-dideazatetrahydrofolic acid) and LY329201 (R)-N-[[5-[2-(2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]pyrimidin-6-yl)ethyl]-2-thienyl]carbonyl]-L-glutamatic acid disodium salt, were investigated in vitro and ex vivo on primary murine peritoneal macrophages and in the RAW macrophage cell line for both purine depletion and inhibition of LPS induced monokine secretion. In vivo efficacy following GARFT inhibition was evaluated in modified rat adjuvant arthritis. RESULTS: LY309886 inhibited purine biosynthesis in the RAW cell line with an EC50 of 90 nM, an effect readily reversible with exogenous hypoxanthine. LY309886 and LY329201 also inhibited LPS induced TNF alpha and MIP1 alpha in these cells and in primary macrophages. A similar effect could be demonstrated ex vivo with mice dosed for two days with 3 mg/kg of LY329201. LY329201 as well as methotrexate demonstrated a dose dependent reduction in both paw and spleen weight and improved joint histology following 2 weeks of dosing in a rat adjuvant arthritis study. CONCLUSION: These results suggest that GARFT inhibitors should be tested in the treatment of rheumatoid arthritis by considering their mechanism of action, here successfully tested on activated macrophages.

Expression of multidrug resistance-associated protein 2 (MRP2) in normal human tissues and carcinomas using tissue microarrays.

AIMS: Using tissue microarrays, this study analysed the expression of the multidrug resistance protein, MRP2, by immunohistochemistry with two different MRP2 antibodies. This is the first study to address the expression of MRP2 in various common human neoplasms. METHODS AND RESULTS: Immunohistochemistry was performed on zinc formalin-fixed tissue to evaluate normal tissues and carcinomas using two antibodies against MRP2 (EAG5, a polyclonal antibody, and M2-lll-6, a monoclonal antibody). Immunostaining was localized in neoplastic cells mainly on the cell membrane with M2-lll-6 and cell membrane and cytoplasm with EAG5. In normal tissues MRP2 was expressed in liver, gastrointestinal tract, and kidney tubular epithelial cells with both antibodies. MRP2 was seen in nine of 22 renal cell carcinomas, eight of 13 gastric carcinomas, 25 of 49 breast carcinomas, 14 of 32 lung carcinomas, 39 of 50 colon carcinomas, and 16 of 17 ovarian carcinomas. There was < 10% variability between the two antibodies. MRP2 expression was highest in moderate to poorly differentiated tumours from colon, lung, gastric, and ovarian carcinomas and in grade 2 and 3 breast and renal carcinomas. CONCLUSION: The expression of MRP2 in many solid human tumours indicates that inherent drug resistance may play an important role as a biomarker for predictive chemotherapy treatment.

Human truncated Smad 6 (Smad 6s) inhibits the BMP pathway in Xenopus laevis.

A previously identified truncated form of the human Smad 6 gene containing a unique 12 amino acid motif at its N-terminus was studied. We have named this truncated form of the gene Smad 6s, for 'short-form', to distinguish it from the full-length form (Smad 6fl). Reverse transcription-polymerase chain reaction and immunohistochemistry revealed that Smad 6s has a unique pattern of expression in human coronary tissue and is upregulated in diseased heart tissue. We used the expression of human Smad 6s in Xenopus laevis as a model system to assess Smad 6s function. Injection of Smad 6fl RNA (4-cell embryos, 2 x ventral) produced tadpoles with partial secondary axes. In contrast, Smad 6s RNA injected in a similar manner produced tadpoles with a severe 'head-only' phenotype with no morphological appearance of a secondary axis. Mutant Smad 6s RNA lacking the unique 12 amino acids at the N-terminus of the Smad 6s isoform produced no embryonic phenotype, suggesting that this region is important in conferring biological activity. Ectodermal explant assays show that Smad 6s has activity consistent with being a BMP antagonist and can synergize with and enhance the activities of the activin and fibroblast growth factor pathways, all of which are novel findings in this study.

Protein kinase C-alpha and -epsilon modulate connexin-43 phosphorylation in human heart.

We have previously demonstrated that protein kinase C (PKC)- alpha expression is significantly elevated in failing human left ventricle, with immunostaining showing increased PKC- alpha localization at the intercalated disks of cardiomyocytes. In the present study we sought to determine, in the failing heart, if PKC- alpha interacted with connexin-43 (Cx-43) both spatially and functionally, and to compare the association of PKC- alpha/Cx-43 with that of PKC- epsilon, a PKC isozyme that does not significantly increase in failing hearts. The possibility of a PKC- alpha or PKC- epsilon/Cx-43 association in non-failing hearts was also investigated. Co-immunoprecipitation of PKC- alpha or PKC- epsilon and Cx-43 in non-failing and failing left ventricle was achieved using antibodies to PKC- alpha or Cx-43. Confocal microscopy confirmed that PKC- alpha distribution within the cardiomyocyte included co-localization with connexin-43 in both failing and non-failing myocardium. In a similar manner, confocal imaging of PKC- epsilon showed cardiomyocyte distribution in both cytosol and membrane, and colocalization of PKC- epsilon with Cx-43. Recombinant PKC- alpha or - epsilon increased PKC activity significantly above endogenous levels in the co-immunoprecipitated Cx-43 complexes (P<0.05). However, phosphorylation of purified human Cx-43 (isolated from failing human left ventricle) by recombinant PKC- alpha or PKC- epsilon resulted in only PKC- epsilon mediated Cx-43 phosphorylation. Thus, in the human heart PKC- alpha, PKC- epsilon, and Cx-43 appear to form a closely associated complex. Whereas only PKC- epsilon directly phosphorylates Cx-43, both PKC isoforms result in increased phosphorylation within the Cx-43 co-immunoprecipitated complex.

Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression.

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancers, particularly in more advanced cancers, suggesting that the AKT/protein kinase B (PKB) kinase, which is negatively regulated by PTEN, may be involved in human prostate cancer progression. We now show that AKT activation and activity are markedly increased in androgen-independent, prostate-specific antigen-positive prostate cancer cells (LNAI cells) established from xenograft tumors of the androgen-dependent LNCaP cell line. These LNAI cells show increased expression of integrin-linked kinase, which is putatively responsible for AKT activation/Ser-473 phosphorylation, as well as for increased phosphorylation of the AKT target protein, BAD. Furthermore, expression of the p27(Kip1) cell cycle regulator was diminished in LNAI cells, consistent with the notion that AKT directly inhibits AFX/Forkhead-mediated transcription of p27(Kip1). To assess directly the impact of increased AKT activity on prostate cancer progression, an activated hAKT1 mutant was overexpressed in LNCaP cells, resulting in a 6-fold increase in xenograft tumor growth. Like LNAI cells, these transfectants showed dramatically reduced p27(Kip1) expression. Together, these data implicate increased AKT activity in prostate tumor progression and androgen independence and suggest that diminished p27(Kip1) expression, which has been repeatedly associated with prostate cancer progression, may be a consequence of increased AKT activity.

Immunohistochemical localization of selected early response genes expressed in trabecular bone of young rats given hPTH 1-34.

Intermittent administration of parathyroid hormone (PTH) increases trabecular bone mass in vivo by stimulating bone formation. To further characterize the cellular and molecular mediators of the anabolic response to PTH, we examined the effect of intermittent synthetic hPTH 1-34 on the expression and localization of selected early response genes, c-fos, c-jun, c-myc, and IL-6 protein, in bone tissue by immunohistochemistry. Young male Sprague-Dawley rats, 70-100 g, were injected s.c. with 8 microg/100 g PTH or vehicle control, once daily for 5 days. Femurs were harvested 1 and 24 hours after the fifth injection, then fixed, decalcified, processed for wax embedding, and sections were immunostained. Early response genes, c-fos, c-jun and IL-6, were strongly expressed in osteoblasts, osteocytes, and megakaryocytes in bones 1 hour after PTH, when compared with vehicle-treated controls or sections from rats, 24 hours after PTH injection. Osteoblasts, osteocytes, and megakaryocytes were also positive for c-myc but the differences in stain intensity between control and treated groups were marginal. Also, scattered islands of hematopoietic cells in the marrow stained intensely for IL-6 by 1 hour after PTH, but the stain intensity decreased to control level 24 hours after the last PTH injection. Scattered islands of hematopoietic cells in the bone marrow stained more strongly for c-fos than either c-jun or c-myc, but neither localization nor stain intensity were regulated by PTH at the time points examined. We conclude that during the immediate early phase of the anabolic response, PTH regulates c-fos, c-jun, and IL-6 expression in osteoblasts, osteocytes, megakaryocytes, and selected bone marrow hematopoietic cells in bone.

Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells.

Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

Heterogeneous loss of connexin43 protein in ischemic dog hearts.

INTRODUCTION: Ischemia causes cell decoupling in the myocardium. Prolonged ischemia activates proteases and causes degradation of structural proteins as well as gap junctions. There is little information about the degradation of gap junction protein during the early time period after acute ischemia. The purpose of the present study was to investigate connexin43 (Cx43) protein degradation and distribution patterns in the canine left ventricular wall during 1 to 6 hours of ischemia. METHODS AND RESULTS: Ischemia of canine left ventricular myocardium was induced by ligation of the left anterior descending coronary artery. Following a period of in situ ischemia of up to 6 hours, samples were harvested, and standard paraffin slides were prepared for Cx43 and wheat germ agglutinin double labeling. Cx43 distribution was visualized by confocal microscopy. In controls, homogeneous distribution of Cx43 staining was determined. Ischemia caused a loss of Cx43 with a heterogeneous pattern by mixing foci of infarcted cells among normal cardiac myocytes. To determine if the changes were induced by heterogeneous reduction in the blood supply, an in vitro ischemic model was studied to induce more homogeneous ischemia. Western blot analysis of these in vitro ischemic tissue samples revealed a reduction of Cx43 protein concentration with a 50% decay time of 4.8 hours. Cx43 dephosphorylation was detected after 1 hour of in vitro ischemia. Heterogeneous loss of Cx43 was found in the in vitro ischemic tissue. There were no significant changes in Cx43 staining density during the first hour of ischemia at a time when dephosphorylation of the protein was observed. After 1 hour of ischemia, Cx43 was reduced at intercalated disk areas, and, after 6 hours, most Cx43 disappeared at intercalated disk areas, while small amounts of Cx43 remained at side-to-side junctions. CONCLUSION: Cx43 undergoes both distribution and concentration changes following acute cardiac ischemia. The loss of Cx43 protein is heterogeneous. Cx43 dephosphorylation occurred within 1 hour following ischemia.

Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart.

BACKGROUND: Increased expression of Ca2+-sensitive protein kinase C (PKC) isoforms may be important markers of heart failure. Our aim was to determine the relative expression of PKC-beta1, -beta2, and -alpha in failed and nonfailed myocardium. METHODS AND RESULTS: Explanted hearts of patients in whom dilated cardiomyopathy or ischemic cardiomyopathy was diagnosed were examined for PKC isoform content by Western blot, immunohistochemistry, enzymatic activity, and in situ hybridization and compared with nonfailed left ventricle. Quantitative immunoblotting revealed significant increases of >40% in PKC-beta1 (P<0.05) and -beta2 (P<0.04) membrane expression in failed hearts compared with nonfailed; PKC-alpha expression was significantly elevated by 70% in membrane fractions (P<0.03). PKC-epsilon expression was not significantly changed. In failed left ventricle, PKC-beta1 and -beta2 immunostaining was intense throughout myocytes, compared with slight, scattered staining in nonfailed myocytes. PKC-alpha immunostaining was also more evident in cardiomyocytes from failed hearts with staining primarily localized to intercalated disks. In situ hybridization revealed increased PKC-beta1 and -beta2 mRNA expression in cardiomyocytes of failed heart tissue. PKC activity was significantly increased in membrane fractions from failed hearts compared with nonfailed (1021+/-189 versus 261+/-89 pmol. mg-1. min-1, P<0.01). LY333531, a selective PKC-beta inhibitor, significantly decreased PKC activity in membrane fractions from failed hearts by 209 pmol. min-1. mg-1 (versus 42.5 pmol. min-1. mg-1 in nonfailed, P<0.04), indicating a greater contribution of PKC-beta to total PKC activity in failed hearts. CONCLUSIONS: In failed human heart, PKC-beta1 and -beta2 expression and contribution to total PKC activity are significantly increased. This may signal a role for Ca2+-sensitive PKC isoforms in cardiac mechanisms involved in heart failure.

The effect of a selective estrogen receptor modulator on the progression of spontaneous autoimmune disease in MRL lpr/lpr mice.

The MRL lpr/lpr mouse strain is an animal model for the autoimmune disorder systemic lupus erythematosus (SLE). Pathologic changes in the mice include a severe proliferative glomerulonephritis, lymph node and spleen enlargement, increase in autoantibody titers, and shortened life spans. In the present investigation, female MRL lpr/lpr mice have been dosed po daily for 7 months with the selective estrogen receptor modulator (SERM) LY139478 (4 mg/kg) or 17alpha-ethinylestradiol (EE2, 1 mg/kg) and compared to vehicle control animals. The LY139478 group had an increase in survival (73% survival at 7 months, P = 0.02) but the EE2-treated animals did not (53% survival at 7 months, P = 0.4) when compared to the control group (32% survival at 7 months). Although there were no reductions in autoantibody levels as determined by anti-DNA antibody ELISA, histological analysis of kidney tissue indicated that both LY139478 and EE2 mitigated the progression of glomerular nephritis which was evident in the controls. In contrast, there were no significant differences in lymph node size although the LY139478 and EE2 groups retained a well-defined sinusoidal region. Finally, flow cytometric analysis documented that thymuses from animals treated for 7 months with LY139478 but not with EE2 contained predominantly CD4+/CD+ T cells consistent with a normal thymic phenotype observed in non-MRL lpr/lpr mouse strains. These studies demonstrate that SERMs may be potentially useful for the treatment of autoimmune disorders.

Pulmonary and mediastinal metastases of a vaccination-site sarcoma in a cat.

Sarcomas at vaccination sites in cats were first reported in 1992. Recent retrospective studies have confirmed an association between these vaccination-site sarcomas (VSS) and feline leukemia virus (FeLV) and/ or rabies vaccines. In most cases, VSS are locally invasive fibrosarcomas that tend to recur but rarely metastasize. We report the mediastinal and pulmonary metastases of a VSS in a FeLV-and feline immunodeficiency virus-negative, 8-year-old, domestic short-haired cat. The primary sarcoma was removed from an interscapular vaccination site and diagnosed as a VSS 3 months prior to radiographic lesions suggestive of pulmonary and mediastinal metastases. At necropsy, there were multiple pulmonary and mediastinal nodules that histologically and ultrastructurally were fibrosarcomas, cytomorphologically similar to the VSS. In addition, immunohistochemical staining patterns of the VSS and metastatic sites were consistent with that described for VSS. Recent reports of pulmonary and mediastinal metastases of interscapular VSS emphasize the importance of early diagnosis and treatment of these tumors.

A family of arginal thrombin inhibitors related to efegatran.

Three new tripeptide arginal thrombin inhibitors were shown to have potent anticoagulant and antithrombotic activity: D-MePhg-Pro-Arg-H (LY287045), D-1-Tiq-Pro-Arg-H (LY294291), and D-MePhe-Pro-Arg-H (Efegatran). Efegatran and the related arginals differ mechanistically from old and from new anticoagulant agents. As illustrated with x-ray diffraction analysis of crystals of the LY294291 complex with human thrombin, the family of arginals binds thrombin with the P3, P2, and P1 residues interacting with the putative S3, S2, and S1 fibrinogen-binding sites. A hemi-acetal bond at Ser 195 was shown to contribute to the tight-binding reversible competitive thrombin inhibition properties observed with the arginal family. Tight-binding Kass values from thrombin inhibition studies correlated with thrombin clottin inhibition potency. The thrombin time (TT) assay was prolonged twofold with 33 nM Efegatran, which demonstrated an apparent Kass value of 0.8 x 10(8) L/mol (for comparison, 17 nM hirudin was required to prolong the TT assay two-fold). There are empirical anticoagulant selectivity differences between Efegatran and hirudin, manifested by large activated partial thromboplastin time (aPTT)/TT effect ratios (30 to 55) found with the arginals, as compared to the small aPTT/TT effect ratio (2 to 3) found with hirudin. The underlying anticoagulant mechanism differences between the arginals and hirudin appear to be confined to the aPTT pathway and, therefore, might involve different effects toward thrombin feedback activation of factor VIII. The arginals did not substantially inhibit other coagulation factor serine proteases. Antithrombotic effects of Efegatran and the arginal family occur at low infusion doses in dogs and appear to correlate with effects on TT without requiring perturbation of the aPTT. Selectivity properties regarding the fibrinolytic enzymes were shown to be important for successful use of the arginals in vivo as adjunctive agents during tissue plasminogen activator (t-PA) thrombolysis. The data suggest that LY287045, LY294291, and Efegatran should be expected to be useful as antithrombotic adjuncts to thrombolytic therapy with t-PA, urokinase, or streptokinase and should be expected to spare endogenous fibrinolysis. Efegatran has been evaluated in phase I clinical studies and is currently under clinical investigation in phase II protocols as a new cardiovascular anticoagulant.

Local delivery of biodegradable microparticles containing colchicine or a colchicine analogue: effects on restenosis and implications for catheter-based drug delivery.

OBJECTIVES: This study sought to evaluate the delivery efficiency, intramural retention and antirestenotic efficacy of soluble colchicine or colchicine analogue delivered into the arterial wall after angioplasty as well as the efficacy of these medications after prolonged local release from biodegradable microparticles. BACKGROUND: Local delivery of pharmacologic agents is a potential treatment for restenosis. However, the delivery efficiency of the technique and the choice of agent to modulate cellular proliferation are unknown. It was hypothesized that restenosis would be unaffected by colchicine or a hydrophobic colchicine analogue with short intramural retention, whereas it would be reduced after prolonged local release. METHODS: Rabbit atherosclerotic femoral arteries underwent angioplasty followed by local delivery. Delivery efficiency and intramural retention of 3H-colchicine were evaluated. The effect of agents in soluble formulation or released from microparticles on angiographic and morphometric restenosis was evaluated at 2 weeks and compared with that in the control groups (angioplasty only and local infusion of carrier solution). RESULTS: Delivery of efficiency was 0.01% and intramural retention < 24 h. Neither soluble colchicine formulation reduced restenosis. Microparticles releasing the colchicine analogue reduced restenosis compared with control and colchicine microparticles but not angioplasty alone (p = 0.002). Delivery outside the artery was observed, and the long-term release of both colchicine resulted in toxicity to the adjacent musculature. CONCLUSIONS: Colchicine or the colchicine analogue did not reduce restenosis, although the long-term local release of the colchicine analogue reduced neointimal proliferation resulting from local delivery. Local delivery of cytotoxic agents with insufficient vascular specificity may be limited by toxicity to adjacent tissues resulting from a larger than expected delivery area and prolonged agent retention.

Ependymoma of the neurohypophysis and hypernatremia in a horse.

A 2-year-old Standardbred gelding was examined because of prolapse of the third eyelid; myoclonus of the muscles of the head, neck, and forelimbs; and persistent tail swishing. The horse had a high plasma sodium concentration but was not drinking water. The hypernatremia could not be corrected by means of IV administration of fluids, and the horse became worse and, 6 days later, died. At necropsy, a tumor was found to be compressing the neurohypophysis and the area in the brain in which the thirst centers are believed to be located. It is believed that hypernatremia in this horse was a result of altered thirst.