Cell Adhesion Molecules Affected by Ionizing Radiation and Estrogen in an Experimental Breast Cancer Model.

Cancer develops in a multi-step process where environmental carcinogenic exposure is a primary etiological component, and where cell-cell communication governs the biological activities of tissues. Identifying the molecular genes that regulate this process is essential to targeting metastatic breast cancer. Ionizing radiation can modify and damage DNA, RNA, and cell membrane components such as lipids and proteins by direct ionization. Comparing differential gene expression can help to determine the effect of radiation and estrogens on cell adhesion. An in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line MCF-10F to low doses of high linear energy transfer α particle radiation and subsequent growth in the presence of 17ß-estradiol. The MCF-10F cell line was analyzed in different stages of transformation that showed gradual phenotypic changes including altered morphology, increase in cell proliferation relative to the control, anchorage-independent growth, and invasive capability before becoming tumorigenic in nude mice. This model was used to determine genes associated with cell adhesion and communication such as E-cadherin, the desmocollin 3, the gap junction protein alpha 1, the Integrin alpha 6, the Integrin beta 6, the Keratin 14, Keratin 16, Keratin 17, Keratin 6B, and the laminin beta 3. Results indicated that most genes had greater expression in the tumorigenic cell line Tumor2 derived from the athymic animal than the Alpha3, a non-tumorigenic cell line exposed only to radiation, indicating that altered expression levels of adhesion molecules depended on estrogen. There is a significant need for experimental model systems that facilitate the study of cell plasticity to assess the importance of estrogens in modulating the biology of cancer cells.

Clinical Findings and Diagnostic Yield of Arrhythmogenic Cardiomyopathy Through Genomic Screening of Pathogenic or Likely Pathogenic Desmosome Gene Variants.

BACKGROUND: Genomic screening holds great promise for presymptomatic identification of hidden disease, and prevention of dramatic events, including sudden cardiac death associated with arrhythmogenic cardiomyopathy (ACM). Herein, we present findings from clinical follow-up of carriers of ACM-associated pathogenic/likely pathogenic desmosome variants ascertained through genomic screening. METHODS: Of 64 548 eligible participants in Geisinger MyCode Genomic Screening and Counseling program (2015-present), 92 individuals (0.14%) identified with pathogenic/likely pathogenic desmosome variants by clinical laboratory testing were referred for evaluation. We reviewed preresult medical history, patient-reported family history, and diagnostic testing results to assess both arrhythmogenic right ventricular cardiomyopathy and left-dominant ACM. RESULTS: One carrier had a prior diagnosis of dilated cardiomyopathy with arrhythmia; no other related diagnoses or diagnostic family history criteria were reported. Fifty-nine carriers (64%) had diagnostic testing in follow-up. Excluding the variant, 21/59 carriers satisfied at least one arrhythmogenic right ventricular cardiomyopathy task force criterion, 11 (52%) of whom harbored DSP variants, but only 5 exhibited multiple criteria. Six (10%) carriers demonstrated evidence of left-dominant ACM, including high rates of atypical late gadolinium enhancement by magnetic resonance imaging and nonsustained ventricular tachycardia. Two individuals received new cardiomyopathy diagnoses and received defibrillators for primary prevention. CONCLUSIONS: Genomic screening for pathogenic/likely pathogenic variants in desmosome genes can uncover both left- and right-dominant ACM. Findings of overt cardiomyopathy were limited but were most common in DSP-variant carriers and notably absent in PKP2-variant carriers. Consideration of the pathogenic/likely pathogenic variant as a major criterion for diagnosis is inappropriate in the setting of genomic screening.

Prevalence and Electronic Health Record-Based Phenotype of Loss-of-Function Genetic Variants in Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Genes.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with variants in desmosome genes. Secondary findings of pathogenic/likely pathogenic variants, primarily loss-of-function (LOF) variants, are recommended for clinical reporting; however, their prevalence and associated phenotype in a general clinical population are not fully characterized. METHODS: From whole-exome sequencing of 61 019 individuals in the DiscovEHR cohort, we screened for putative loss-of-function variants in PKP2, DSC2, DSG2, and DSP. We evaluated measures from prior clinical ECG and echocardiograms, manually over-read to evaluate ARVC diagnostic criteria, and performed a PheWAS (phenome-wide association study). Finally, we estimated expected penetrance using Bayesian inference. RESULTS: One hundred forty individuals (0.23%; 59±18 years old at last encounter; 33% male) had an ARVC variant (G+). None had an existing diagnosis of ARVC in the electronic health record, nor significant differences in prior ECG or echocardiogram findings compared with matched controls without variants. Several G+ individuals satisfied major repolarization (n=4) and ventricular function (n=5) criteria, but this prevalence matched controls. PheWAS showed no significant associations of other heart disease diagnoses. Combining our best genetic and disease prevalence estimates yields an estimated penetrance of 6.0%. CONCLUSIONS: The prevalence of ARVC loss-of-function variants is ≈1:435 in a general clinical population of predominantly European descent, but with limited electronic health record-based evidence of phenotypic association in our population, consistent with a low penetrance estimate. Prospective deep phenotyping and longitudinal follow-up of a large sequenced cohort is needed to determine the true clinical relevance of an incidentally identified ARVC loss-of-function variant.

Differential expression of cell adhesion molecules in an ionizing radiation-induced breast cancer model system.

Cell-cell adhesion is mediated by members of the cadherin-catenin system and among them E-cadherin and ß-catenin are important adhesion molecules for epithelial cell function and preservation of tissue integrity. To investigate the importance of cell adhesion molecules in breast carcinogenesis, we developed an in vitro breast cancer model system wherein immortalized human breast epithelial cell line, MCF-10F, was malignantly transformed by exposure to low doses of high linear energy transfer (LET) α particle radiation (150 keV/µm) and subsequent growth in the presence or absence of 17ß-estradiol. This model consisted of human breast epithelial cells in different stages of transformation: i) parental cell line MCF-10F; ii) MCF-l0F continuously grown with estradiol at 10(-8) (Estrogen); iii) a non-malignant cell line (Alpha3); and iv) a malignant and tumorigenic cell line (Alpha5) and the Tumor2 cell line derived from the nude mouse xenograft of the Alpha5 cell line. Expression levels of important cell adhesion molecules such as α-catenin, ß-catenin, γ-catenin, E-cadherin and integrin were found to be higher at the protein level in the Alpha5 and Tumor2 cell lines relative to these levels in the non-tumorigenic MCF-10F, Estrogen and Alpha3 cell lines. In corroboration, cDNA expression analysis revealed elevated levels of genes involved in the cell adhesion function [E-cadherin, integrin ß6 and desmocollin3 (DSc3)] in the Alpha5 and Tumor2 cell lines relative to the levels in the MCF-10F, Estrogen and Alpha3 cell lines. Collectively, our results suggest that cell adhesion molecules are expressed at higher levels in malignantly transformed breast epithelial cells relative to levels in non-malignant cells. However, reduced levels of adhesion molecules observed in the mouse xenograft-derived Tumor 2 cell line compared to the pre-tumorigenic Alpha5 cell line suggests that the altered expression levels of adhesion molecules depend on the tumor tissue microenvironment.

Comparative study of autoantigen profile between Colombian and Brazilian types of endemic pemphigus foliaceus by various biochemical and molecular biological techniques.

BACKGROUND: Besides Brazilian endemic pemphigus foliaceus (EPF), we have described another focus of EPF in Colombia. Our previous study suggested that Colombian EPF seemed to react various plakin family proteins, such as envoplakin, periplakin and BP230. OBJECTIVE: To further characterize the Colombian EPF and study the difference from Brazilian EPF, we examined the antigen profile of the two types of EPF. METHODS AND RESULTS: Immunoblotting using normal human epidermal extracts revealed that 38% Colombian EPF sera and 25% Brazilian EPF sera showed IgG antibodies reactive with desmoglein (Dsg) 1, pemphigus foliaceus antigen. The sera of both types of EPF showed protein bands co-migrating with plakin family proteins, particularly periplakin. Immunoblotting analyses using recombinant proteins of various domains of envoplakin, periplakin and BP230 revealed that a considerable number of Colombian EPF sera reacted with recombinant proteins of periplakin, while only few Brazilian sera reacted with some of the recombinant proteins of any plakins. Enzyme-linked immunosorbent assay (ELISA) for Dsg1 and Dsg3 showed that Dsg1 was reacted by almost all sera of both types of EPF. However, unexpectedly, while none of Colombian EPF sera reacted with Dsg3, about half of Brazilian EPF sera reacted with Dsg3. CONCLUSION: These results suggested that the Colombian EPF is basically similar to Brazilian EPF in terms that major antigen is Dsg1, but there were some different antigen profiles between the two types of EPF.

Two Brazilian cases of IgA pemphigus.

IgA pemphigus is a rare, neutrophilic, acantholytic skin disorder with approximately 70 cases described in the literature. We report two patients with the subcorneal pustular dermatosis (SPD) type of IgA pemphigus. Initially, both patients were misdiagnosed as subcorneal pustular dermatosis of Sneddon and Wilkinson. The correct diagnosis was only made after detecting intercellular IgA depositions in the epidermis by direct immunofluorescence. Immunoblotting (IB) of normal human epidermal extracts, performed on both sera, was negative for Dsg 1, Dsg 3, BP 230, BP 180, 210 kDa envoplakin, and 190 kDa periplakin. ELISA for desmogleins (Dsg 1 and Dsg 3) showed that neither of the cases had IgA antibodies to Dsg. The c-DNA transfection test for desmocollins (Dsc) revealed that the IgA antibodies of both patients reacted with desmocollin 1. This result supports the hypothesis that the autoantigen in SPD type IgA pemphigus is desmocollin 1.

Desmocollins I and II are recognized by certain sera from patients with various types of pemphigus, particularly Brazilian pemphigus foliaceus.

Recently, it has been shown that desmoglein, pemphigus foliaceus target antigen, and a 130-kD pemphigus vulgaris antigen belong to the cadherin family of cell adhesion molecules. We tried to determine whether desmocollins I/II, other cadherin-like transmembranous glycoproteins present in desmosomes, are also recognized by pemphigus autoantibodies of the IgG class. We examined 16 pemphigus vulgaris sera, 15 pemphigus foliaceus sera, 15 Brazilian pemphigus foliaceus sera, five bullous pemphigoid sera, and 65 normal sera. Four (25%) pemphigus vulgaris sera, one (7%) pemphigus foliaceus serum, eight (53%) Brazilian pemphigus foliaceus sera, and three (5%) normal sera reacted with desmocollins I/II on immunoblots of bovine desmosome preparation. The affinity-purified desmocollins I/II pemphigus autoantibodies were shown to bind the epidermal cell surface by indirect immunofluorescence. Immunoblot analysis revealed one pemphigus vulgaris serum, one Brazilian pemphigus foliaceus serum, and one normal serum recognizing a recombinant protein produced by a desmocollin cDNA clone. Moreover, immunoblot analysis of reactivity of a Brazilian pemphigus foliaceus serum with recombinant proteins produced by deletion mutants of the desmocollin cDNA clone showed that the extracellular portion of desmocollin is immunogenic in this pemphigus patient. We conclude that desmocollins I/II are recognized by certain sera from patients with various types of pemphigus, particularly Brazilian pemphigus foliaceus. However, the significance of this reactivity remains to be defined.