An immortalized human cell line bearing a PRKAR1A-inactivating mutation: effects of overexpression of the wild-type Allele and other protein kinase A subunits

CONTEXT: Inactivating mutations of PRKAR1A, the regulatory subunit type 1A (RIalpha) of protein kinase A (PKA), are associated with tumor formation. OBJECTIVE: Our objective was to evaluate the role of PKA isozymes on proliferation and cell cycle. METHODS: A cell line with RIalpha haploinsufficiency due to an inactivating PRKAR1A mutation (IVS2+1 G-->A) was transfected with constructs encoding PKA subunits. Genetics, PKA subunit mRNA and protein expression and proliferation, aneuploidy, and cell cycle status were assessed. To identify factors that mediate PKA-associated cell cycle changes, we studied E2F and cyclins expression in transfected cells and E2F's role by small interfering RNA; we also assessed cAMP levels and baseline and stimulated cAMP signaling in transfected cells. RESULTS: Introduction of PKA subunits led to changes in proliferation and cell cycle: a decrease in aneuploidy and G(2)/M for the PRKAR1A-transfected cells and an increase in S phase and aneuploidy for cells transfected with PRKAR2B, a known PRKAR1A mutant (RIalphaP), and the PKA catalytic subunit. There were alterations in cAMP levels, PKA subunit expression, cyclins, and E2F factors; E2F1 was shown to possibly mediate PKA effects on cell cycle by small interfering RNA studies. cAMP levels and constitutive and stimulated cAMP signaling were altered in transfected cells. CONCLUSION: This is the first immortalized cell line with a naturally occurring PRKAR1A-inactivating mutation that is associated in vivo with tumor formation. PKA isozyme balance is critical for the control of cAMP signaling and related cell cycle and proliferation changes. Finally, E2F1 may be a factor that mediates dysregulated PKA's effects on the cell cycle.     

Impact of organisational change on mental health: a systematic review

Although limited evidence is available, organisational change is often cited as the cause of mental health problems. This paper provides an overview of the current literature regarding the impact of organisational change on mental health. A systematic search in PUBMED, PsychInfo and Web of Knowledge combining MeSH search terms for exposure and outcome. The criterion for inclusion was original data on exposure to organisational change with mental health problems as outcome. Both cross-sectional and longitudinal studies were included. We found in 11 out of 17 studies, an association between organisational change and elevated risk of mental health problems was observed, with a less provident association in the longitudinal studies. Based on the current research, this review cannot provide sufficient evidence of an association between organisational change and elevated risk of mental health problems. More studies of long-term effects are required including relevant analyses of confounders.     

Detection of somatic beta-catenin mutations in primary pigmented nodular adrenocortical disease (PPNAD)

Background Primary pigmented nodular adrenocortical disease (PPNAD) leads to Cushing syndrome (CS) and is often associated with Carney complex (CNC). Genetic alterations of the type 1‐α regulatory subunit of cAMP‐dependent protein kinase A (PRKAR1A) and phosphodiesterase 11A4 (PDE11A) genes have been found in PPNAD. Recent studies have demonstrated that β‐catenin mutations are frequent in adrenocortical adenomas and carcinomas and that the Wnt‐signalling pathway is involved in PPNAD tumorigenesis. We hypothesized that adrenocortical adenomas that form in the context of PPNAD may harbour β‐catenin mutations. Methods We studied 18 patients with CS secondary to PPNAD who were screened for germline PRKAR1A and PDE11A mutations. Tumor DNA was extracted from pigmented adrenocortical adenoma and nodular adrenal hyperplasia. Mutation analysis of exons 3 and 5 of β‐catenin was performed using polymerase chain reaction and direct sequencing. Sections from formalin‐fixed, paraffin‐embedded tumour samples were studied by immunohistochemistry with an antibody against β‐catenin. Results Nine patients were carrying germline PRKAR1A mutations and one patient had a PDE11A mutation. We found somatic β‐catenin mutations in 2 of 18 patients (11%). In both cases, the mutations occurred in relatively large adenomas that had formed in the background of PPNAD. Tumor DNA analysis revealed a heterozygous ACC‐to‐GCC missense mutation in codon 41 (T41A) and a TCT‐to‐CCT missense mutation in codon 45 (S45P) of exon 3 of the β‐catenin gene that was confirmed at the cDNA level. There were no alterations in the DNA of PPNAD‐adjacent tissues and lymphocytes from the patients, indicating somatic events. Immunohistochemistry showed nuclear accumulation of β‐catenin in more than 90% of cells in adenomatous tissue whereas no nuclear immunoreactivity was detected in adjacent PPNAD nodular cells. Nuclear translocation of β‐catenin protein in the PPNAD adenoma suggests activation of the Wnt–β‐catenin pathway in PPNAD. Conclusions We report, for the first time, β‐catenin mutations in adenomas associated with PPNAD, further implicating Wnt–β‐catenin signalling in tumorigenesis linked to bilateral adrenal hyperplasias.     

Etiology and site of temporal lobe epilepsy influence postictal cytokine release

Inflammatory mechanisms are involved in the pathogenesis of epilepsy. Vice versa, immune functions are regulated by the brain. We measured postictal changes in serum levels of the immuno-modulating cytokines IL-1β, IL-6 and TNFα in patients with well-defined temporal lobe epilepsy (TLE) and determined modifying factors. Serum levels of IL-1β, IL-6 and TNFα were quantified by ELISA at baseline as well as immediately, 1 h and 24 h after a complex partial (CPS) or secondary generalized tonic–clonic seizure (GTCS) during video-EEG monitoring in 25 patients suffering from temporal epilepsy. IL-6 increased by 51% immediately after the seizure (p < 0.01) and remained elevated for 24 h. This increase lacked in patients with hippocampal sclerosis (HS; n = 16, mean increase 28%, p > 0.5, vs. 112%, p < 0.01 in patients without HS). IL-6 levels were higher after right-sided seizures as compared to left-sided seizures 24 h after the seizure (8.7 pg/mL vs. 3.4 pg/mL, p < 0.05). In patients taking valproate (VPA, n = 9), the levels of IL-1β were higher as compared to patients not treated with VPA. The results suggest a relationship between the cytokine system and characteristics of TLE such as side and pathology.     

Determination of the epigenetic effects of ochratoxin in a human kidney and a rat liver epithelial cell line.

Epidemiological studies have implicated ochratoxin A (OTA), a fungal metabolic-contaminant of animal and human food sources, in Balkan Endemic Nephropathy and renal tumors. Many environmental toxicants operate through nongenotoxic mechanisms that epigenetically control gene expression leading to a diseased state. Gap junctional intercellular communication (GJIC) plays a central role in the epigenetic control of genes in which alteration of normal GJIC has been implicated in many human pathologies, including cancer, teratogenesis, reproductive dysfunction and peripheral neuropathies. The cell proliferative stages of human diseases, such as cancer, also involves the induction of signal transduction pathways controlling the mitogenic steps, in which the mitogen activated protein kinases (MAPK), such as extracellular receptor kinase (ERK) and p38, are central to mitogenesis. We therefore determined the effects of OTA on GJIC and MAPK in a human kidney and rat liver epithelial cell line. OTA reversibly inhibited GJIC at noncytotoxic doses in the rat liver but not the human kidney cell line. Similarly, OTA was also a strong activator of MAPK, ERK and p38, in the rat liver cells but only weakly activated ERK and had no affect on p38 in the human kidney cell line. Another hallmark of human diseases is an abnormal alteration of apoptosis, also known as programmed cell death. We used our myc-transfected cell line, which exhibits higher levels of apoptosis, to test the effects of OTA on apoptosis. OTA greatly induced apoptosis in this cell line, which is contrary to the effects of most tumor promoters. In summary, OTA exhibits tumor promoting properties in the liver, but the effects of OTA on the human kidney epithelial cells suggested a lack of tumorigenic activity assuming that these epithelial cells, like the rat liver epithelial cells, are a primary target for carcinogens. These results also indicate that the nephrotoxicity of OTA either does not involve GJIC, assuming these epithelial cells play a vital role in kidney physiology, or that a more differentiated kidney cell type is the target for OTA toxicity, of which the role of GJIC remains unknown.