Plasma proteins rather than amino acid chelates are the direct sources of copper for mammalian cells. In continuing studies on the mechanisms by which albumin and transcuprein deliver copper and the potential involvement of CTR1, rates of uptake from these proteins and Cu-histidine were compared in cells with/without CTR1 knockdown or knockout. siRNA knocked down expression of CTR1 mRNA 60-85% in human mammary epithelial and hepatic cell models, but this had little or no effect on uptake of 1 μM Cu(II) attached to pure human albumin or alpha-2-macroglobulin. Mouse embryonic fibroblasts that did/did not express Ctr1 took up Cu(II) bound to albumin about as readily as from the histidine complex at physiological concentrations and by a single saturable process. Uptake from mouse albumin achieved a 2-4-fold higher Vmax (with a lower Km) than from heterologous human albumin. Maximum uptake rates from Cu(I)-histidine were >12-fold higher (with higher Km) than for Cu(II), suggesting mediation by a reductase. The presence of cell surface Cu(II) and Fe(III) reductase activity responding only slightly to dehydroascorbate was verified. Excess Fe(III) inhibited uptake from albumin-Cu(II). Ag(I) also inhibited, but kinetics were not or un-competitive. In general there was little difference in rates/kinetics of uptake in the Ctr1+/+ and -/- cells. Endocytosis was not involved. We conclude that plasma proteins deliver Cu(II) to homologous cells with greater efficiency than ionic copper at physiological concentrations, probably through the mediation of a Steap Cu(II)-reductase, and confirm the existence of an additional copper uptake system in mammalian cells.
CTR1 is the only specific Cu uptake transporter so far identified in mammalian cells. However in some cell types, Ag(I) (which inhibits CTR1) does not impede Cu uptake from plasma proteins or the histidine complex. To further test the importance of CTR1, we measured rates of uptake in cells with/without knockdown and knockout. siRNA knockdown (65–85%) of CTR1 mRNA made no difference to rates of Cu(II) uptake from human albumin and alpha2macroglobulin by human mammary epithelial and hepatic cell lines. Uptake of Cu from Cu(I)‐histidine or Cu(II)‐mouse albumin by mouse embryonic fibroblasts was only ~15% less in Ctr1‐null versus wild type cells (kindly provided by Dennis Thiele). Maximum rates of uptake from the his complex were ~10× greater for Cu(I) than Cu(II). The kinetics of Cu(II) uptake from mouse albumin were closer to those of Cu(I)‐his (Vmax 14 pmol/min/mg cell protein; Km 1 uM), while those from human albumin were similar to Cu(II)‐his (Vmax 4–6, Km 2–4.5). Cell surface Cu(II) and Fe(III) reductase activity was detected; but Fe(III) failed to inhibit uptake from Cu(II)‐albumin. In the Ctr1 null cells, there also was some inhibition by Ag(I). It is concluded that mammalian cells contain at least one additional Ag(I)‐inhibitable Cu transporter, that prefers Cu(I), probably receiving it upon reduction of Cu(II) by a cell surface reductase. Supported by HHMI and USPHS Grant RO1 HD46949.
Introduction: Large-scale chromosomal deletions frequently occur in the progression of head and neck squamous cell carcinoma (HNSCC). Many studies have linked these deletions with particular patient outcomes. However, their precise contributions to tumorigenesis have yet to be elucidated. Here, we introduce an in vitro model for studying large-scale chromosomal deletions to establish their role as driver or passenger events in HNSCC progression. Methods: CRISPR/Cas9 vectors coding for guide RNAs (gRNAs) that flank two of the commonly deleted regions in HNSCC tumors were transfected into primary human keratinocytes. Eighteen different combinations of gRNAs were utilized to create deletions of various sizes at two different genomic loci. Genomic DNA (gDNA) was harvested 24 hours post-transfection and analyzed via endpoint PCR and Sanger sequencing to assess for the presence of the deletions. Edited cells were further sorted via fluorescence-activated cell sorting (FACS) and the efficiency of the deletions were analyzed via performing TaqMan copy number assay qPCR on extracted gDNA. Subsequently, the invasive properties of the genome-edited cells were then investigated using organotypic cultures, a near-physiological three-dimensional model system for studying squamous epithelial biology. Results: The feasibility of performing large-scale deletions was confirmed via endpoint PCR and Sanger sequencing, in which all 18 gRNA combinations created successful deletions. We further sought to create a pure population of the cells harboring the deletions by isolating the edited cells via FACS. TaqMan copy number assay qPCR on gDNA harvested from these cells indicated a 10-45% reduction in the copy number of loci of interest. Genome-edited keratinocytes containing deletions at two of the most commonly deleted sites in HNSCC tumors were used for organotypic assays, in which the preliminary results suggested that deletion of these sites can lead to a gain of invasive behavior. Conclusion: Chromosomal deletions occur at a high frequency in HNSCC tumors. However, there is no established model to determine whether these deletions exist as driver or passenger events. Here, we demonstrate the introduction of successful large-scale deletions in a primary human cell culture system that can be further utilized to study the role of these events in HNSCC tumorigenesis. Furthermore, we show that two of the most common deletions in HNSCC may act as driver events in tumor progression. Support: This study was supported by Program in Breakthrough Biological Research, UCSF School of Dentistry OHNRG pilot grant, and an R01 grant (DE029890-01 - Defining the Mutational pathogenesis of Oral Preneoplasia). Citation Format: Ramin Farhad, Vicente Planells Palop, Aaron Tward. Functional characterization of chromosomal deletions in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2504.
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