No evidence for oncogenic mutations in guanine nucleotide-binding proteins of human adrenocortical neoplasms

G-Proteins are membrane-bound heterotrimeric polypeptides that couple receptor signals to second messenger systems such as cAMP. Recently, point mutations at 2 codons of the highly preserved alpha-chain of Gs, the adenyl cyclase-stimulating G-protein, were found in GH-secreting pituitary tumors. These mutations resulted in constitutively activated Gs alpha and high intracellular cAMP levels. In addition, point mutations at similar codons of a different G-protein, G(i) alpha 2, were reported in adrenocortical neoplasms, suggesting a potential role of this isoform in the genesis of these tumors. We reevaluated the frequency of constitutively activating point mutations in the alpha- chain of the stimulatory (Gs alpha) and inhibitory (G(i) alpha 2) G- proteins in human adrenocortical tumors. Seven adrenocortical carcinomas, 2 human adrenocortical tumor cell lines, and 11 adrenocortical adenomas were studied. Genomic DNA was purified from either frozen tumor tissue or paraffin-embedded sections. Using specific primers and the polymerase chain reaction, DNA fragments surrounding codons 201 and 227 (Gs alpha) and 179 and 205 (G(i) alpha 2) were amplified and visualized on a 2% agarose gel. In a second asymmetric polymerase chain reaction, using nested primers, single stranded DNA was generated using 1-10 microL of the initial amplification mixture and directly sequenced using the dideoxy chain termination method of Sanger. We found no mutations at codons 201, 227 and 179, 205 of Gs alpha and G(i) alpha 2, respectively, in the tumors studied. We conclude that previously identified oncogenic point mutations in the stimulatory and inhibitory alpha-chain of G-proteins do not appear to be present at high frequency in adrenal neoplasms. Thus, the mechanism(s) of tumorigenesis in these tumors is different from that in GH-secreting adenomas and may involve oncogenic mutations of other cell constituents

P53 mutations in human adrenocortical neoplasms

The mechanisms of tumorigenesis of adrenocortical neoplasms have not been elucidated as yet. However, loss of heterozygosity at chromosomal locus 17p has been consistently observed in adrenocortical cancer. p53 is a recessive tumor suppressor gene located on chromosome 17p. Mutations in the p53 gene play an important role in the tumorigenesis of diverse types of human neoplasms including breast and colon cancers. More than 90% of all mutations discovered in such tumors have been detected in 4 hot spot areas that lie between exons 5 and 8. In contrast to wild-type p53, mutant p53 accumulates intracellularly and can be easily detected by immunohistochemistry. We therefore investigated the frequency of p53 mutations in human adrenocortical neoplasms using molecular biology and immunohistochemistry techniques. Five patients with adrenocortical adenomas (5 female; ages 39-72 yr), 11 patients with adrenocortical carcinomas (8 female, 3 male; ages 15- 50 yr), and two adrenocortical tumor cell lines were studied. After DNA extraction from frozen tumor tissue or paraffin-embedded material, exons 5 through 8 were amplified using the polymerase chain reaction and directly sequenced by the dideoxy termination method. Immunohistochemistry was performed on paraffin-embedded tumor specimens obtained during adrenalectomy using a monoclonal antibody reacting with both wild-type and mutant p53. Prevalence of mutations was adenomas, 0/5, carcinomas, 3/11, and adrenocortical cell lines, 2/2. Single point mutations were detected in 3 cases (exons 5, 6, and 7, respectively), and rearrangements of exon 7/8 and 8 were found in 2 cases. Immunohistochemistry detected strong nuclear and/or cytoplasmic p53 immunoreactivity in all adrenocortical carcinomas with point mutations of the p53 gene but not in adenomas and carcinomas with the wild-type sequence or with deletion/rearrangement of the p53 gene. We conclude that p53 plays a role in the tumorigenesis of adrenocortical carcinomas but is of less importance to benign adenomas

Epicurean Stability (eustatheia): A Philosophical Approach of Stress Management

Epicurus used an empirical and sensualistic approach to knowledge, creating a consistent, naturalistic, pragmatic and consequentialistic philosophy. The scientific observations of the last centuries have confirmed the basic principles of Epicurean physics, as well the psychotherapeutic approach of Epicurean ethics, which fits human nature. We know from the work “On Frank Criticism” of Epicurean philosopher Philodemus of Gadara, that the teaching methodology of Epicureans included psychoeducational counseling through therapeutic criticism based on friendly freedom of speech and aiming at τῆς ψυχῆς θεραπείαν (psychotherapy) and at knowledge of maintaining mental health and well-being. The Epicureans called εὐστάθεια (eustatheia, “stability”) the psychosomatic balance (τὸ τῆς σαρκὸς καὶ ψυχῆς εὐσταθὲς κατάστημα), which today we call homeostasis (ὁμοιόστασις), and considered it the basis of true happiness. They recognized empirically the stress that disturbed psychosomatic homeostasis as an agitation of the psyche or a painful feeling of the body and used a number of mental and affective techniques (including the tetrapharmakos) to manage stress at its onset, so that it does not evolve into the particularly troublesome conditions of anxiety and/or depression, which may become chronic psychosomatic disorders with significant social consequences. The article discusses the relation of the main ethical teachings of Epicurus with the biological basis of human brain functions and with the management of stress by cognitive and behavioral psychotherapy

Clonal Composition of Human Adrenocortical Neoplasms

The mechanisms of tumorigenesis of adrenocortical neoplasms are still not understood. Tumor formation may be the result of spontaneous transformation of adrenocortical cells by somatic mutations. Another factor stimulating adrenocortical cell growth and potentially associated with formation of adrenal adenomas and, less frequently, carcinomas is the chronic elevation of proopiomelanocortin-derived peptides in diseases like ACTH-dependent Cushing's syndrome and congenital adrenal hyperplasia. To further investigate the pathogenesis of adrenocortical neoplasms, we studied the clonal composition of such tumors using X-chromosome inactivation analysis of the highly polymorphic region Xcen-Xp11.4 with the hybridization probe M27ß, which maps to a variable number of tandem repeats on the X-chromsome. In addition, polymerase chain reaction amplification of a phosphoglycerokinase gene polymorphism was performed. After DNA extraction from tumorous adrenal tissue and normal leukocytes in parallel, the active X-chromosome of each sample was digested with the methylation-sensitive restriction enzyme HpaII. A second digestion with an appropriate restriction enzyme revealed the polymorphism of the region Xcen-Xp11.4 and the phosphoglycerokinase locus. Whereas in normal polyclonal tissue both the paternal and maternal alleles are detected, a monoclonal tumor shows only one of the parental alleles. A total of 21 female patients with adrenal lesions were analyzed; 17 turned out to be heterozygous for at least one of the loci. Our results were as follows: diffuse (n = 4) and nodular (n = 1) adrenal hyperplasia in patients with ACTH-dependent Cushing's syndrome, polyclonal pattern; adrenocortical adenomas (n = 8), monoclonal (n = 7), as well as polyclonal (n = 1); adrenal carcinomas (n = 3), monoclonal pattern. One metastasis of an adrenocortical carcinoma showed a pattern most likely due to tumor-associated loss of methylation. In the special case of a patient with bilateral ACTH-independent macronodular hyperplasia, diffuse hyperplastic areas and a small nodule showed a polyclonal pattern, whereas a large nodule was monoclonal. We conclude that most adrenal adenomas and carcinomas are monoclonal, whereas diffuse and nodular adrenal hyperplasias are polyclonal. The clonal composition of ACTH-independent massive macronodular hyperplasia seems to be heterogeneous, consisting of polyclonal and monoclonal areas

Philosophical Management of Stress based on Science and Epicurean Pragmatism: A Pilot Study

In the first months of the COVID-19 pandemic, we created and implemented from November 2020 to February 2021 a monthly educational pilot program of philosophical management of stress based on Science, Humanism and Epicurean Pragmatism, which was offered to employees of 26 municipalities in the Prefecture of Attica, Greece. The program named “Philosophical Distress Management Operation System” (Philo.Di.M.O.S.) is novel and unique in its kind, as it combines a certain Greek philosophical tradition (Epicurean) that concurs with modern scientific knowledge. The program was designed to be implemented in a period of crisis; therefore, it used a fast-paced, easy to learn and practice philosophical approach to stress management, based on cognitive psychotherapy. The philosophical approach to stress management has the advantage that it can be offered to most people, regardless of age and educational level. The pilot program was effective in achieving its objectives, shown by statistical comparisons of the trainees’ responses to anonymous questionnaires before and after the month-long training. The successful Philo.Di.M.O.S. program, thus, based on a solid scientific and philosophical basis, offers a paradigm of stress management during crises and could be useful in Greece and internationally