Protease Activity in Protein-Free Ns0 Myeloma Cell Cultures

Abstract

Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.

Cathepsin L in Normal and Pathological Bone Remodeling

“…In vitro studies show that tumor cells and osteoclasts, once activated by pro-inflammatory cytokines with bone resorption activity, secrete Cathepsin L, which participates in degrading bone matrix, thus allowing the homing of metastatic cells into the bone tissue [119-122, 127, 130-135]. These findings are consistent with recent in vitro observations, which demonstrate that the expression levels of Cathepsin L in tumor tissue are increased by proinflammatory cytokines with bone resorption activity such as IL-1b, IL-6 or TNF-a [67,68,[120][121][122][123][124][125]. Furthermore, Cathepsin L may indirectly promote bone degradation by triggering the activation of latent precursor forms of other proteolytic enzymes, such as MMPs, uPA and heparanase, which are known to foster metastatic cascade and bone remodeling processes [8,43,45,119,136].…”

“…In support of this hypothesis, experimental and clinical observations show that the expression levels of this endopeptidase are altered in some primary bone tumors [81,82,[119][120][121][122][123], in bone metastasis [81,82,119,120] and in tumors that preferentially metastasize to the bone [118,119,[125][126][127][128][129][130][131][132]. For instance, Park et al [81] report that Cathepsin L is expressed in 100% of metastatic bone tumors and only in 50% of primary human bone tumors.…”

Proliferation of NS0 cells in protein-free medium: The role of cell-derived proteins, known growth factors and cellular receptors

“…Negative effects of ubiquitin on certain haematopoietic cells have been shown by Daino et al (2000). The presence of the cystein protease inhibitor cystatin C and the serine protease inhibitor SLPI in NS0 CM has been addressed in a previous publication together with the finding that NS0 cells also secrete several proteases (Spens and Häggström, 2005b). A group of serine proteases, active at culture pH, was shown to affect NS0 cell proliferation negatively.…”

Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography

“…There are several proteases that degrade gelatin (denatured collagen), which include the two matrix metalloproteases MMP-2 and MMP-9 [15]. Other enzymes are serine proteases like trypsin, plasmin and matriptase, as well as cysteine proteases such as cathepsin L [16,17,18,19]. In this protocol we focus on proteases that function around a neutral pH, but the protocol can be slightly changed for the detection of proteases with an optimal activity at slightly acidic pH (see Note 1).…”