Anti-NEFH monoclonal antibody, clone 4H4

Neurofilament heavy polypeptide (NEFH) has recently been identified as a candidate DNA hypermethylated gene within the functional breast cancer hypermethylome. NEFH exists in a complex with neurofilament medium polypeptide (NEFM) and neurofilament light polypeptide (NEFL) to form neurofilaments, which are structural components of the cytoskeleton in mature neurons. Recent studies reported the deregulation of these proteins in several malignancies, suggesting that neurofilaments may have a role in other cell types as well. Using a comprehensive approach, we studied the epigenetic inactivation of neurofilament genes in breast cancer and the functional significance of this event. We report that DNA methylation-associated silencing of NEFH, NEFL, and NEFM in breast cancer is frequent, cancer-specific, and correlates with clinical features of disease progression. DNA methylation-mediated inactivation of these genes occurs also in multiple other cancer histologies including pancreas, gastric, and colon. Restoration of NEFH function, the major subunit of the neurofilament complex, reduces proliferation and growth of breast cancer cells and arrests them in Go/G1 phase of the cell cycle along with a reduction in migration and invasion. These findings suggest that DNA methylation-mediated silencing of the neurofilament genes NEFH, NEFM, and NEFL are frequent events that may contribute to the progression of breast cancer and possibly other malignancies.

Purpose: To determine whether the human Muller cell line Moorfields/Institute of Ophthalmology-Muller 1 (MIO-M1) expresses opsins. Methods: The gene expression of opsins was determined by reverse-transcription PCR (RT-PCR). The presence of opsin proteins was determined by western blotting and immunocytochemistry. The light sensitivity of the cells was examined with imaging experiments using the calcium-sensitive dye Fluo-4. Results: MIO-M1 cells express glial (glutamine synthase [GLUL], vimentin [VIM], glial fibrillary acidic protein [GFAP], cellular retinaldehyde-binding protein [RLBP1], glial high-affinity glutamate transporter [SLCA1], aquaporin-4 [AQP4], inwardly rectifying potassium channel Kir4.1 [Kir4.1]), neuronal (Thy-1 cell surface antigen [THY1], heavy neurofilament polypeptide [NEFH], microtubule-associated protein 2 [MAP2], neurogenic differentiation 1 [NEUROD1], neuronal nuclei [NEUN]), and neural progenitor markers (Nestin [NES], paired-type homeobox transcription factor [PAX6], neurogenic locus notch homolog 1 [NOTCH1]). The cells contain mRNA for the following opsins: blue opsin (OPN1SW), rhodopsin (OPN2), panopsin (OPN3), melanopsin (OPN4), neuropsin (OPN5), and peropsin (RRH), as well as for the transducins (guanine nucleotide binding protein [GNAZ], alpha transducing activity polypeptide 1 [GNAT1], alpha transducing activity polypeptide 2 [GNAT2]). The presence of blue opsin and melanopsin was confirmed with immunocytochemistry and western blotting. The immunoreactivity and mRNA of red-green opsin were found in some but not all cultures, while the immunoreactivity for rhodopsin was absent in all cultures investigated. Repetitive stimulation with 480 nm light evoked slow and fast transient calcium responses in the majority of cells investigated, while irradiation with 600 nm light was ineffective. Conclusions: The human Muller cell line MIO-M1 expresses opsins. This suggests immortalized Muller cells could be used as a cellular source to produce human opsins for their potential application as therapeutic agents in patients with retinitis pigmentosa.