Fibroblast lysosomes

Pisoni, R. L., and Thoene, J. G. (1989). Detection and characterization of a nucleoside transport system in human fibroblast lysosomes. J. Biol. Chem. 264, 4850-4856. 

Warburton, M. J., and Wynn, C. H., The effect of intralysosomal sucrose storage on the turnover of hamster fibroblast lysosomal and Golgi-apparatus enzymes. Biochem. J. 158,401-407 (1976). 

One limitation of enzyme replacement therapy is the targeting of enzyme proteins to appropriate sites of substrate accumulation. Administration of a cholesterol esterase conjugated to albumin results in the degradation of pathologic cholesterol ester accumulations within the lysosomes of fibroblasts from a patient with cholesterol ester storage disease (246). 

Man 6-P receptors, located in the Golgi apparatus, bind the Man 6-P residue of these enzymes and direct them to the lysosomes. Fibroblasts from patients with I-cell disease (see below) are severely deficient in the activity of the GIcNAc phosphotransferase. 

As indicated above, Man 6-P serves as a chemical marker to target certain lysosomal enzymes to that organelle. Analysis of cultured fibroblasts derived from patients with I-cell (inclusion cell) disease played a large part in revealing the above role of Man 6-P. I-cell disease is an uncommon condition characterized by severe progressive psychomotor retardation and a variety of physical signs, with death often occurring in the first decade. Cultured cells from patients with I-cell disease were found to lack almost all of the normal lysosomal enzymes the lysosomes thus accumulate many different... 

The diagnosis of lysosomal disorders is usually based on enzymatic assays in white blood cells or cultured skin fibroblasts [ 1 ]. Molecular studies required for identification of carriers. Initial diagnosis of peroxisomal disorders... 

The test is based on an in vitro assay of the uptake of the dye, neutral red (NR), in Balb/c 3T3 fibroblasts. It was developed to detect the phototoxicity induced by the combined interaction of the test substance and light of the wavelength range from 315 to 400 nm, the so-called UVA. The cytotoxicity is evaluated in the presence (+UVA) or absence (-UVA) of UVA light exposure, after application of a nontoxic dose of the compound. The cytotoxicological impact is assessed via the inhibition of the fibroblasts to take up the vital dye NR (NR is a weak cationic dye, penetrating easily into the cell membrane by a nonionic diffusion and accumulates in the lysosomes) one day after the initial treatment. Normally, healthy cells may incorporate and bind NR. Alterations of the cell surface or the lysosomal membranes, however, lead to a decreased uptake and binding of the dye. 

Brunk, U.T., Dalen, H., Roberg, K.., and Hellquist, H.B., 1997, Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts. Free Radio. 

Assays are frequently needed to detect marked and acute cytotoxicity that may confound the interpretation of cell-based efficacy assays. Neutral red uptake is one of the most commonly used cytotoxicity assays and is used in the regulatory phototoxicity assay on NT3 fibroblasts [13]. It has been show to be more sensitive than assays for mitochondrial reductive capacity such as the tetrazolium reductase assays, ATP depletion assays, or for cell permeabilization or mpture such as dye uptake or lactate dehydrogenase leakage. Lysosomes take up, protonate and trap neutral red when cellular ATP production is sufficient to maintain pH gradients. 

The lysosomal disorder SASD is characterized by accumulation of the free acid monosaccharide sialic acid in the lysosomal compartment of the cell. Diagnosis is based on the demonstration of abnormal excretion of free, not OGS-bound sialic acid in urine, coupled with accumulation of free sialic acid in cultured fibroblasts, and on microscopic evidence of vacuoles (increased and swollen lysosomes filled with light electron-lucent material in skin biopsy and peripheral blood lymphocytes). The inheritance is autosomal recessive. There are different clinical forms of this disorder an adult form, called Salla disease (SD) or Finnish sialuria (OMIM 604369) infantile SASD (ISSD OMIM 269920) and an intermediate form, severe Salla disease [3,16]. 

Explain why fibroblasts from a patient with I-cell disease secrete lysosomal enzymes when grown in tissue culture. 

Receptor-mediated uptake of LDL by human skin fibroblasts. Specific LDL receptors are located in coated regions of the plasma membrane. LDL binding results in uptake by endocytosis and formation of a coated vesicle. This vesicle fuses with a lysosome containing many hydrolytic enzymes that degrade the lipoprotein, releasing cholesterol. 

The metabolism of GSLs has been studied in cultured human fibroblasts from normal subjects, patients with lipid storage diseases, and those with FH. The content of the GSLs, as well as activities of the biosynthetic enzymes, the glycosyltransferases and the lysosomal GSL hydrolases,have been studied. Complex gangliosides, such as M1, GDla, have been found in this cell system to serve as receptors for cholera toxin and thyrotropin, respectively (24-26). More recently, GT1 and GDla have been postulated to be receptors for fibronectin in cultured fibroblasts... 

Glycosphinqolipid Hydrolases. The deficiency of lysosomal glycosylhydrolases has been shown in a number of lipid storage diseases (as summarized in Table II) using cultured fibroblasts (29). ... 

Lysosomal Glycosyl Hydrolase Deficiency and the Accumulation of Glycosphingolipids in Fibroblasts in "Glycolipidosis" ... 

Specifically, the data reviewed in Chapters 12 and 13 indicate that LCM are rapidly removed from the circulation by the tumor the maximum accumulation of LCM in the tumor area occurs within the first 30 min after administration (ref. 531). These rapid kinetics for LCM uptake are quite consistent with the well-documented kinetics long-known for the LDL receptor-mediated endocytic pathway (ref. 616). For example, Goldstein et al. (ref. 643) reported that LDL-ferritin bound to coated pits at 4°C is rapidly internalized when fibroblasts in tissue culture are warmed to 37°C. In this uptake process, the coated pits invaginate to form coated endocytic vesicles. After 5 to 10 min at 37°C, LDL-ferritin is observed in lysosomes as the result of their fusion with the incoming coated vesicles (ref. 643). The rapid sequence of events visualized in electron micrographs precisely parallels biochemically-derived data on the rapid uptake and degradation of radiolabeled LDL (ref. 644,645). 

The first adenoviral (Ad)-mediated gene delivery studies for GSDII were performed in GSDII patient fibroblasts, myoblasts, and myotubes (Nicolino et al., 1998 Pauly et al., 1998, 2001). Several important findings came from these early studies including the ability to achieve overexpression of GAA from Ad vectors of up to 19-20-fold over untreated normal cells (Nicolino et al., 1998 Pauly et al., 1998), localization of the Ad-delivered GAA protein to the lysosomes (Pauly et al., 1998, 2001), clearance of accumulated glycogen in treated cells (Nicolino et al., 1998 Pauly et al., 1998), secretion of the 110 kDA precursor form into the culture media (Nicolino et al., 1998 Pauly et al., 1998), and M6P receptor-mediated uptake of GAA secreted from transduced cells by GAA-deficient cells (Nicolino et al., 1998 Pauly et al., 1998). 

Pauly, D. F., Johns, D. C., Matelis, L. A., Lawrence, J. H., Byrne, B. J. and Kessler, P. D. (1998). Complete correction of acid alpha-glucosidase deficiency in Pompe disease fibroblasts in vitro, and lysosomally targeted expression in neonatal rat cardiac and skeletal muscle. Gene Ther. 5, 473-480. 

In recent years, as interest in the properties of biological membranes has increased, researchers have used lectins to study the role of the binding of multivalent ligands to the plasms membrane in the induction of specific biological responses. This report will detail the use of lectins to study the structure of the carbohydrate moiety of lymphocyte surface bound H-2 glycoproteins. Furthermore, it will describe a novel lectin-induced perturbation of the metabolism of in vitro cultured fibroblasts which results in the massive accumulation of lysosomes in these cells. This response is greatly reduced in cells which have been transformed by oncogenic viruses. 

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