Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C

Coulibaly, Sogue; Schwihla, Herwig; Abrahamson, Magnus; Albini, Adriana; Cerni, Christa; Clark, Jason L.; Ng, Ken M.; Katunuma, Nobuhiko; Schlappack, O; Glössl, Josef; Mach, Lukas

doi:10.1002/(sici)1097-0215(19991112)83:4<526::aid-ijc15>3.3.co;2-d

Cited by 15 publications

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“…In cystatin C transfected B16 melanoma cells, the overexpression of inhibitor resulted in the inhibition of melanoma cell mobility and of the ability to penetrate artificial matrices by about 50% (Sexton and Cox, 1997), as well as in the suppression of metastasis by at least 90%, compared to controls (Cox et al, 1999). Similarly, the overproduction of active recombinant cystatin C resulted in a pronounced reduction in Matrigel invasion of murine squamous carcinoma cells (Coulibaly et al, 1999). In human glioblastoma cells, inverse correlation between cystatin C and tumour grade was observed (Konduri et al, 2002).…”

Section: Discussionmentioning

confidence: 96%

Cysteine proteinase inhibitor cystatin C in squamous cell carcinoma of the head and neck: relation to prognosis

et al. 2004

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To determine the role of the cysteine proteinase inhibitor cystatin C in the invasive behavior of squamous cell carcinoma of the head and neck (SCCHN), Cystatin C protein level was measured in 82 pairs of primary tumour tissue and adjacent noncancerous mucosa, using the enzyme-linked immunosorbent assay. The median level of cystatin C in tumour tissue was 1.18 times lower than that in corresponding mucosa (P ¼ 0.031). In normal mucosa samples, the cystatin C level was influenced by the site of sampling: it was lower in nonlaryngeal tissue samples (oral cavity, oro-or hypopharynx) than in laryngeal samples (P ¼ 0.004). The tumour cystatin C level correlated inversely with pN-stage (P ¼ 0.047), whereas a trend of lower cystatin C levels was observed in the group with extranodal tumour extension compared to those with no extranodal spread (P ¼ 0.069). In univariate analysis, the patients with low tumour cystatin C levels exhibited poor disease-free survival (DFS, P ¼ 0.013) and disease-specific survival (DSS, P ¼ 0.013). In multivariate analysis, the most powerful predictor of survival was pN-stage (DFS: P ¼ 0.040, HR 2.78; DSS: P ¼ 0.011, HR 4.36,), followed by the cystatin C level (DFS: P ¼ 0.043, HR 0.22; DSS: P ¼ 0.067, HR 0.25). When comparing the prognostic strength of cystatin C to that of stefin A, another cysteine proteinase inhibitor, which emerged as the most significant prognosticator for survival in our previous study analysing the same cohort of patients, stefin A proved to be significantly more reliable predictor for both DFS and DSS than cystatin C. Our results indicate that cystatin C is implicated in the invasive behavior of SCCHN, and that there are variations in regulation of proteolytic pathways under nonmalignant conditions, inherent to individual subsites inside the upper aerodigestive tract. The correlation between high cystatin C levels and improved survival concurs with the concept of the protective role of high levels of cysteine proteinase inhibitors in tissue homogenates that has been previously suggested by the survival results in breast and lung carcinoma as well as SCCHN.

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Section: Discussionmentioning

confidence: 96%

Cysteine proteinase inhibitor cystatin C in squamous cell carcinoma of the head and neck: relation to prognosis

et al. 2004

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“…29 HeLa human cervical carcinoma cells and NIH 3T3 murine fibroblasts (both from the American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's Modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin and 100 lg/ml streptomycin. All tissue culture reagents were purchased from Invitrogen (Carlsbad, CA).…”

Section: Cell Culturementioning

confidence: 99%

“…Samples were denatured for 5 min at 65°C (M6P/IGF2R) or 95°C (other proteins) prior to electrophoresis and immunoblotting analysis as reported. 29,34 Densitometric analysis of immunoblots was done as described. 30 Immobilization and biotinylation of M6P/IGF2R ligands Hansenula holstii NRRL Y-2448 phosphomannan (a kind gift of Dr. Morey E. Slodki, US Department of Agriculture, Peoria, IL) was rehydrated in 100 ml of 1% KCl at 4°C for 16 hr.…”

Section: Immunoblotting Analysismentioning

confidence: 99%

“…29 Several purified growth factors including IGF-II are also able to induce SCC-VII migration. We have now found that the migratory capacity of SCC-VII/ IGF2R-1 and SCC-VII/IGF2R-2 cells is less than 50% of that of parental and mock-transfected SCC-VII cells irrespective of the chemoattractant used.…”

Section: Expression Of M6p/igf2r Decreases Invasion Of Scc-vii Cells mentioning

confidence: 99%

“…Furthermore, SCC-VII cells lack mature lysosomes and store their acid hydrolases instead in compartments reminiscent of late endosomes. 10,29,30 In this study, we have generated SCC-VII lines stably reconstituted with human M6P/IGF2R to directly assess the impact of this receptor on the tumourigenicity and invasiveness of squamous cell carcinoma cells. …”

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The mannose 6‐phosphate/insulin‐like growth factor II receptor restricts the tumourigenicity and invasiveness of squamous cell carcinoma cells

Probst¹,

Puxbaum²,

Svoboda³

et al. 2009

Intl Journal of Cancer

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The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) mediates biosynthetic sorting and endocytosis of various factors that impinge on the proliferation, migration and invasiveness of tumour cells. The gene encoding M6P/IGF2R is frequently lost or mutated in a wide range of malignant tumours including squamous cell carcinomas. We have previously shown that M6P/IGF2R-deficient SCC-VII murine squamous cell carcinoma cells secrete large amounts of pro-invasive lysosomal proteinases. Furthermore, the formation of mature lysosomes is impaired in SCC-VII cells. To assess the link between M6P/ IGF2R status and tumour invasion, we have now generated SCC-VII lines stably transfected with human M6P/IGF2R cDNA. Reconstitution of functional M6P/IGF2R expression in SCC-VII cells strongly improves the intracellular retention of lysosomal proteinases and restores the formation of mature lysosomes. In addition, the presence of heterologous M6P/IGF2R compromises the growth of SCC-VII cells both in vitro and in vivo. Remarkably, M6P/IGF2R expression also reduces the invasive capacity of SCC-VII cells in response to various chemoattractants. These results indicate that the M6P/IGF2R status influences the metastatic propensity of squamous cell carcinomas. ' 2008 Wiley-Liss, Inc.Key words: cathepsin; lysosome; proteolysis; squamous cell carcinoma; tumour invasion A key requirement for metastatic cancer cell invasion is the penetration of extracellular matrix (ECM) barriers. This process involves the degradation of different ECM proteins and proteoglycans. 1,2 Various proteinases have been implicated in ECM degradation associated with tumour invasion and metastasis, including urokinase-type plasminogen activator, matrix metalloproteinases and cathepsins. [3][4][5] The involvement of the latter enzymes in ECM proteolysis is perplexing, since cathepsins are normally localized in lysosomes. However, tumour cells often secrete significant amounts of these proteinases into the pericellular fluid, as first observed for cathepsin B. 6 As typical for lysosomal enzymes, the N-glycan moieties of cathepsins are modified during their biosynthesis with mannose 6-phosphate (M6P) residues which permit interaction with the main lysosomal sorting receptors, the 300-kDa mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) and the 46-kDa mannose 6-phosphate receptor (MPR46). 7 Evidence has been provided that excessive secretion of cathepsins by tumour cells may be due to transformation-induced changes to the M6P receptor pathway. [8][9][10] Lysosomal sorting via M6P/IGF2R is generally far more efficient than by MPR46, demonstrating that the former is the main lysosomal targeting receptor in mammalian cells. 11,12 However, M6P/IGF2R also binds a variety of other factors that impinge on the proliferation, migration and invasiveness of tumour cells, including insulin-like growth factor II (IGF-II), 13 transforming growth factor b, 14 urokinase-type plasminogen activator receptor 15 and plasminogen. 16 Hence, it is o...

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New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations

et al. 2007

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Background: Cathepsins are endosomal/lysosomal proteases that play important roles in regulating cell physiological processes in cardiovascular, neurological, musculoskeletal, and immunological systems. Pathophysiological processes are often associated with a change in cathepsin expression and activity, leading to the possibility of using cathepsins as disease markers for diagnosis and prognosis. Methods: We describe a new assay utilizing an argon laser flow cytometer to measure activities of cysteine cathepsins B, L, and S in live cells using cell permeable fluorogenic cresyl violet-conjugated peptides as selective substrates. Substrate concentration dependency and time kinetics studies were performed. The activity assay was combined with immunofluorescence staining to detect cell lineage-specific molecules and assess cathepsin activities in a heterogeneous cell population.

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Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C

Cited by 15 publications

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Cysteine proteinase inhibitor cystatin C in squamous cell carcinoma of the head and neck: relation to prognosis

Cysteine proteinase inhibitor cystatin C in squamous cell carcinoma of the head and neck: relation to prognosis

The mannose 6‐phosphate/insulin‐like growth factor II receptor restricts the tumourigenicity and invasiveness of squamous cell carcinoma cells

New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations

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