Identification of a lung cancer cell line deficient in atg7-dependent autophagy

<div><p>ABSTRACT</p><p>Autophagy is a major cellular process for bulk degradation of proteins and organelles in order to maintain metabolic homeostasis, and it represents an emerging target area for cancer. Initially proposed to be a cancer-restricting process for tumor initiation, recent studies suggest that autophagy can also promote cell survival in established tumors. <i>ATG7</i> is an essential autophagy gene that encodes the E1 enzyme necessary for the lipidation of the LC3 family of ubiquitin-like proteins and autophagosome formation. In this study we identified a rare case of a cancer cell line, H1650 lung adenocarcinoma, which has lost ATG7 expression due to a focal biallelic deletion within the <i>ATG7</i> locus. These cells displayed no evidence of ATG7 pathway activity; however, reconstituting the cells with wild-type ATG7 restored both LC3 lipidation and downstream autophagic consumption of autophagy substrates such as the SQSTM1/p62 protein. We characterized several phenotypes reported to be influenced by autophagy, and observed an ATG7-dependent increase in cell growth and clearance of proteasome-inhibitor induced protein aggregates. Cellular changes in mitochondrial metabolism or response to nutrient starvation were unaffected by ATG7 expression. In addition, parental H1650 cells that lacked ATG7 were still able to consume autophagy substrates SQSTM1, NBR1 and TAX1BP1 via a bafilomycin A₁-sensitive pathway, suggesting that these proteins were not exclusively degraded by autophagy. Overall, these findings highlight a unique outlier instance of complete loss of ATG7-dependent autophagy in a cancer cell line. The H1650 cell line may be a useful system for future studies to further understand the role of autophagy in tumorigenesis and potential redundant pathways that allow cells to circumvent the loss of ATG7-dependent autophagy in cancer.</p></div

Sequence analysis of beta-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma treated with bortezomib or dexamethasone

Variations within proteasome beta (PSMB) genes, which encode the beta subunits of the 20S proteasome, may affect proteasome function, assembly, and/or binding of proteasome inhibitors. To investigate the potential association between PSMB gene variants and treatment-emergent resistance to bortezomib and/or long-term outcomes, in the present study, PSMB gene sequence variation was characterized in tumor DNA samples from patients who participated in the phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) study of bortezomib versus high-dose dexamethasone for treatment of relapsed multiple myeloma. Twelve new PSMB variants were identified. No associations were found between PSMB single nucleotide polymorphism genotype frequency and clinical response to bortezomib or dexamethasone treatment or between PSMB single nucleotide polymorphism allelic frequency and pooled overall survival or time to progression. Although specific PSMB5 variants have been identified previously in preclinical models of bortezomib resistance, these variants were not detected in patient tumor samples collected after clinical relapse from bortezomib, which suggests that alternative mechanisms underlie bortezomib insensitivity. This study is registered at www.clinicaltrials.gov as NCT00048230. (Blood. 2012;120(23):4513-4516

Polycystic kidney disease: the complete structure of the PKD1 gene and its protein

Mutations in the PKD1 gene are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). Other PKD1-like loci on chromosome 16 are approximately 97% identical to PKD1. To determine the authentic PKD1 sequence, we obtained the genomic sequence of the PKD1 locus and assembled a PKD1 transcript from the sequence of 46 exons. The 14.5 kb PKD1 transcript encodes a 4304 amino acid protein that has a novel domain architecture. The amino-terminal half of the protein consists of a mosaic of previously described domains, including leucine-rich repeats flanked by characteristic cysteine-rich structures, LDL-A and C-type lectin domains, and 14 units of a novel 80 amino acid domain. The presence of these domains suggests that the PKD1 protein is involved in adhesive protein-protein and protein-carbohydrate interactions in the extracellular compartment. We propose a hypothesis that links the predicted properties of the protein with the diverse phenotypic features of ADPKD

Sequence analysis of β-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma treated with bortezomib or dexamethasone

Variations within proteasome β (PSMB) genes, which encode the β subunits of the 20S proteasome