A quantitative assay for collagen synthesis in microwell fibroblast cultures

Webster, David; Harvey, W. Proctor

doi:10.1016/0003-2697(79)90576-1

Cited by 112 publications

(51 citation statements)

References 6 publications

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“…18,31 Briefly, at the end of the [ 14 C]Glycine pulse, 2 l 2% bovine serum albumin (BSA) was added, followed by addition of 1/3 the volume of 2 mg/ml pepsin in 2 M acetic acid, and the cultures were incubated for 4 hr at room temperature (RT). Quadruplicate samples were pooled and carrier collagen type I (Vitrogen-100; Collagen, Palo Alto, CA) added to a final concentration of 0.2 mg/ml.…”

Section: Quantification Of Collagen Synthesismentioning

confidence: 99%

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Collagen type I expression in experimental anaplastic thyroid carcinoma: Regulation and relevance for tumorigenicity

Dahlman

Lammerts

Bergström

et al. 2001

Intl Journal of Cancer

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Fibrosis in solid malignancies plays a significant role in tumor pathophysiology. Potential mechanisms for collagen type I deposition in anaplastic thyroid carcinoma (ATC) were investigated using 6 characterized ATC cell lines. Three of these cell lines, which produced collagen type I, had, as a group, a poor tumorigenicity when inoculated in athymic mice. This group of cells generated tumors in 4 of 24 injected animals (17%). Pro-␣1(I) collagen mRNA-expressing carcinoma and stromal cells were interdispersed in the tumors generated by these ATC cells. By contrast, the 3 noncollagen-producing ATC cell lines were all tumorigenic with a tumor take of 60% in the whole group. In the latter tumors, pro-␣1(I) collagen mRNA-expressing cells were confined to the stromal compartment, well delineated from carcinoma cell islets. To study the influence of ATC cells on collagen type I synthesis by fibroblasts, we used AG 1518 diploid human fibroblasts cultured on poly-(2-hydroxyethyl methacrylate) (poly[HEMA])-coated plates. This culture condition allows the study of the effect of collagen mRNA translation in the regulation of collagen type I synthesis. Conditioned media from the 6 ATC cell lines did not influence collagen synthesis. The ATC cell line KAT-4 stimulated fibroblast synthesis of collagen type I when the two cell types were cocultured on poly[HEMA]-coated substrates. Specific inhibitors of PDGF and TGF-␤ reduced the KAT 4 carcinoma cell-induced stimulation of collagen type I synthesis. Our data suggest that collagen type I production by carcinoma cells correlates negatively with tumorigenicity and that the formation of a well-defined stroma is of importance for tumor growth. Furthermore, our data suggest that tumor cells are able to stimulate collagen mRNA translation in stromal fibroblasts in direct cell-cell contact by, at least in part, transferring PDGF or TGF-␤.

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Section: Quantification Of Collagen Synthesismentioning

confidence: 99%

“…Pepsin-resistant collagen was precipitated as described. 18,31 Metabolically labelled collagen was analyzed by SDS-PAGE fluorography and quantified by Phosphor Imager (Fuji).…”

Section: Quantification Of Collagen Synthesismentioning

confidence: 99%

Collagen type I expression in experimental anaplastic thyroid carcinoma: Regulation and relevance for tumorigenicity

Dahlman

Lammerts

Bergström

et al. 2001

Intl Journal of Cancer

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“…6) by their co-elution and co-migration, respectively, with standard collagens from rat skin or radioactivelylabelled human dermal fibroblast cultures. Recovery of radioactive collagen from CMC chromatography was consistently > 90%0, due to pre-treatment of the CMC with an excess of rat-skin collagen (Webster & Harvey, 1979).…”

Section: Resultsmentioning

confidence: 95%

“…Identification of radioactively-labelled collagens by carboxymethyl cellulose (CMC) chromatography was performed as described previously (Webster & Harvey, 1979). Approximately 80,000 dpm of each 3H-labelled collagen sample was dissolved with 2 mg of carrier acid-soluble rat-skin collagen in 0-06M acetate buffer, pH 4-8, containing IM urea, and applied to a 6-0 x 1 5cm column of CM 52 (Whatman) maintained at 450C.…”

Section: Methodsmentioning

confidence: 99%

“…Samples of 70,000 dpm were dissolved in the above buffer containing IM urea, 2% glycerol and a trace of bromophenol blue as marker dye. Electrophoresis was performed in the buffer described above at 1 mamp/gel and the gels were subsequently sliced into 1mm-thick discs before alkaline hydrolysis and measurement of radioactivity (Webster & Harvey, 1979).…”

Section: Methodsmentioning

confidence: 99%

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A biochemical and immunohistological study of collagen synthesis in Ewing's tumour

Harvey¹,

Squier²,

Duance³

et al. 1982

Br J Cancer

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Summary.-The synthesis and localization of collagen have been studied on material from a total of 16 primary Ewing's tumours. The predominant collagen extracted from the tissues and synthesized in short-term cultures was type I. The proportion of type III collagen was relatively small and variable (0-8%) in the direct tumour extracts, but a higher proportion (29-38% of the total collagens) was synthesized in culture. Immunofluorescence studies showed that positive staining for all types of collagen tested (types I, III, IV and V) was restricted to stroma; there was no evidence of collagen either within the tumour cells or in their pericellular matrix, a finding endorsed by negative staining for reticulin in the same areas. The absence of any evidence for type IV or V collagen synthesis by Ewing's cells argues against an endothelial origin for the tumour, and indicates that collagen analysis is unlikely to be of value in the diagnosis of this particular sarcoma.

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Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared withL-ascorbate in human intestinal smooth muscle cells

et al. 1999

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The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.

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A quantitative assay for collagen synthesis in microwell fibroblast cultures

Cited by 112 publications

References 6 publications

Collagen type I expression in experimental anaplastic thyroid carcinoma: Regulation and relevance for tumorigenicity

Collagen type I expression in experimental anaplastic thyroid carcinoma: Regulation and relevance for tumorigenicity

A biochemical and immunohistological study of collagen synthesis in Ewing's tumour

Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared withL-ascorbate in human intestinal smooth muscle cells

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