Changes in the amount and distribution of prosomal subunits during the differentiation of U937 myeloid cells: high expression of p23K

Abstract

Prosomes (Proteasomes/Multicatalytic proteinase (MCP)-complexes) are protein particles built of 28 subunits in variable composition, having proteinase activity. We have studied the changes in prosomal subunits p29K, p31K and the highly expressed p23K during the differentiation of U937 cells. Control cells had little prosomal subunit p31K in the cytoplasm, while p29K antigen was detected in both the nucleus and cytoplasm; more p23K antigen was found in the cytoplasm than in the nucleus. Flow cytometry demonstrated a biphasic intracellular decrease in prosomes during differentiation induced by phorbol-myristic-acetate (PMA) and retinoic acid plus 1,25-dihydroxycholecalciferol (RA + VD). p23K and p29K decreased both in the cytoplasm and the nucleus of differentiated cells, though the p23K antigen was concentrated near vesicles and the plasma membrane in PMA-induced cells. The p31K antigens disappeared from RA + VD-induced cells, while in PMA-induced cells, cytoplasmic labelling was unchanged and nuclear labelling was increased. Small amounts of prosomal proteins p23K and p29K were found on the outer membrane of un-induced cells. While there was no labelling on the outer membrane of RA + VD-induced cells, p23K protein increased on the plasma membrane of PMA-induced cells. The prosome-like particle protein p21K was not present to any significant extent in the intracellular compartment of control or induced cells; however, p21K was detected on the outer surface of control cells and was increased only in PMA-induced cells. The culture medium of control and induced cells contained no p21K, p23K, p29K or p31K. RA + VD seemed to induce a general decrease of prosomal subunits within the cells and at the outer surface, whereas PMA caused a migration toward the plasma membrane and an increase at the outer surface. These changes in the distribution and type of prosomes in RA + VD- and PMA-induced cells indicate that prosomes may play a part in differentiation, especially p23K which is the most highly expressed protein among those studied and presents the more important changes.

High plasma proteasome levels are detected in patients with metastatic malignant melanoma

“…Given the wide range of proteins directed towards degradation by proteasomes, including proteins controlling the cell cycle, it has been demonstrated, as expected, that a high proteasome cell level is observed in immature stages of cell differentiation and neoplastic processes 7–9 . Proteasome expression has been demonstrated in the skin, 10 and its expression and activity decrease with age together with a modification in subunit composition, this being correlated with tissue accumulation of oxidatively modified and ubiquitinated proteins 11–13 .…”

Plasma proteasome level is a potential marker in patients with solid tumors and hemopoietic malignancies

“…Anomalies in the ubiquitin‐proteasome pathway have been described in various pathologic settings, including malignant disease,18 and proteasome inhibitors can induce tumor regression in animal models 19. Along with others, we have published modifications in the intracellular expression and subunit composition of proteasome in leukemic cells4–8 as well as abnormal proteasome levels in tumor and stromal cells in some patients of breast carcinoma 20. Moreover, gene expression of the proteasome subunit iota is increased in patients with ALL and decreased in patients with AML 21…”

“…In 1990, Kumatori et al4 first demonstrated an abnormally high expression level of proteasomes in human leukemic cells. We subsequently published findings that, in some examples of neoplastic differentiation, the amount and subunit composition of cell proteasomes indeed varied 5–8. Proteasomes can be detected and measured on the cell surface of lymphocytes9 and in cell culture supernatants or patient serum 10.…”

Extracellular, circulating proteasomes and ubiquitin — Incidence and relevance

“…In vitro proteasomes have been shown to bind to phospholipid monolayers with a preferred orientation [23] and in vivo they may even be partially buried within the membrane because a certain fraction of ER-associated proteasomes was found to be resistant to degradation by trypsin [19]. The presence of proteasomes at the surface of cell membrane was reported by Bureau and coworkers, who detected proteasomes at the cell surface of a non-permeabilized human leukaemic cell line (U937) and of human T and B lymphocytes by use of proteasome subunit-specific antibodies and flow cytometry [24,25]. According to these investigations the pattern of proteasomal epitopes detectable at the surface of the cells changed depending on their stage of differentiation after treatment with phorbol-myristate ester, retinoic acid or vitamin D3 and their cluster of differentiation, respectively.…”

“…According to these investigations the pattern of proteasomal epitopes detectable at the surface of the cells changed depending on their stage of differentiation after treatment with phorbol-myristate ester, retinoic acid or vitamin D3 and their cluster of differentiation, respectively. The authors could not detect proteasomes in the culture medium of the leukaemic cells [25], but nevertheless speculated that the enzymes are released somehow into the extracellular medium and may function as paracrine factors [24].…”