Arja Jukkola

Aberrant type I and type III collagen gene expression in human breast cancer in vivo

Increased synthesis and degradation of extracellular matrix components are associated with breast cancer development. This study evaluated type I and type III procollagen mRNA expression and the corresponding protein synthesis and maturation, as well as the tissue distribution of these collagens, in benign breast lesions, infiltrating ductal carcinomas, and their metastases by in situ hybridization and immunohistochemistry. In the benign lesions, the type I and type III collagen bundles were regularly organized and the expression of the corresponding mRNA was weak, indicating a relatively slow collagen turnover. In the malignant tumours, increased expression of type I and type III procollagen mRNAs was observed in the fibroblastic cells of the stroma; the malignant epithelial cells did not participate. The staining of corresponding newly‐synthesized pN‐collagens showed aberrant bundles in the invasive front of the malignant tumours. Newly‐synthesized type I and type III procollagens were occasionally observed in fibroblastic cells, particularly in grade 2 and grade 3 tumours. Metastases of breast carcinoma resembled poorly differentiated primary tumours with respect to their collagen synthesis and deposition. The increased synthesis of fibrillar type I and type III procollagens may serve as a pathway for tumour invasion. The enhanced synthesis is associated with the formation of aberrant collagen bundles, which may be more readily degradable and may thus facilitate breast tumour invasion. Copyright

Type I procollagen synthesis is regulated by steroids and related hormones in human osteosarcoma cells

Changes in the synthesis of type I collagen, the major extracellular matrix component of skin and bone, are associated with normal growth, tissue repair processes, and several pathological conditions. Expression of the COL 1A1 gene is regulated by transcriptional and post‐transcriptional mechanisms. However, the hormonal regulation of type I collagen synthesis in human bone has not been well characterized. We have studied the influence of calcitriol, dexamethasone, retinoic acid, and estradiol on the COL 1A1 gene expression by determining the secretion of the C‐terminal propeptide (PICP) and the levels of α1(I) procollagen mRNA in cultured human MG‐63 and SaOs‐2 osteoblast‐like osteosarcoma cells. Similar experiments were also performed with respect to expression of the nuclear proto‐oncogenes, c‐fos and c‐jun, in MG‐63 cells.

Increased expression of collagen types I and III in human skin as a consequence of radiotherapy

To study the mechanisms of irradiation-induced fibrosis, the expression of types I and III collagen was analysed in radiotherapy-treated human skin. The subjects were ten randomly chosen women who had been treated for breast cancer with surgery and radiotherapy. The subjects ranged in age from 42 to 68xa0years (mean 53xa0years) and the time from treatment ranged from 7 to 94xa0months. The irradiated skin was compared with a corresponding healthy skin area in the same subject. Suction blisters were induced on both skin areas. The aminoterminal propeptides of types I and III collagen (PINP and PIIINP), which reflect actual in vivo skin collagen synthesis, were determined in the suction blister fluid using radioimmunoassays. mRNA of types I and III collagen were determined in skin specimens using a nonradioactive in situ hybridization (ISH) technique. Immunohistochemical staining for PINP was also performed. The level of PINP in suction blister fluid was increased more than threefold and the level of PIIINP more than twofold in irradiated skin compared to control skin. The number of cells containing type I and type III collagen mRNA was increased in the upper dermis of irradiated skin. Immunohistochemical staining showed the amount of PINP-positive fibroblasts to be increased in irradiated skin. We conclude that skin collagen gene expression is increased as a result of irradiation and this leads to fibrosis and thickening of the dermis.

Modulation of skin collagen metabolism by irradiation: collagen synthesis is increased in irradiated human skin

Radiation‐induced fibrosis is a common side‐effect of cancer treatment. The pathophysiological events leading to fibrosis are not known in detail. We analysed the effect of therapeutic irradiation on human skin collagen synthesis, skin thickness, gelatinases and their inhibitors. Twenty randomly chosen women who had been treated for breast cancer with surgery and radiation therapy participated in the study. In each patient, the irradiated skin area was compared with a corresponding non‐treated skin area. Suction blister fluid (SBF) and serum samples were analysed for the aminoterminal propeptides of type I and type III procollagens (PINP and PIIINP), tissue inhibitors of matrix metalloproteinases (MMPs) 1 and 2 (TIMP‐1 and TIMP‐2) and MMP‐9 and MMP‐2/TIMP‐2 complex. Skin biopsies were analysed for PINP and immunohistochemical staining was used for PIIINP. In irradiated skin, PINP, PIIINP, TIMP‐1 and MMP‐2/TIMP‐2 complex levels in SBF and the number of PINP‐positive fibroblasts in tissue sections were significantly higher in comparison with non‐treated skin. The levels of TIMP‐2 in irradiated and non‐irradiated skin were similar. MMP‐9 could not be detected in SBF with the assay used. The serum levels of MMP‐9 were higher in the treated subjects than the reference values. The serum values of PINP, PIIINP, TIMP‐1, TIMP‐2 and MMP‐2/TIMP‐2 complex were not significantly affected. These results indicate increased local collagen synthesis and accumulation of connective tissue in irradiated skin. The marked upregulation of collagen synthesis as a result of irradiation offers a possibility to treat this complication with compounds such as topical steroids which downregulate collagen synthesis.

Type I collagen turnover and cross-linking are increased in irradiated skin of breast cancer patients

BACKGROUND AND PURPOSEnThe effects of radiation therapy on the turnover and structure of type I collagen were studied in irradiated and contralateral skin of 18 breast cancer patients without clinically evident fibrosis.nnnMATERIALS AND METHODSnThe rates of on-going type I collagen synthesis and degradation were assessed by the aminoterminal propeptide of type I procollagen (PINP) and by two different assays (ICTP and SP4) for the carboxyterminal telopeptide of type I collagen in the soluble tissue extracts, respectively. Also, TIMP-1, TIMP-2 and the MMP-2/TIMP-2 complex were measured in the tissue extracts. Insoluble skin matrices, containing the cross-linked type I collagen fibres, were heat-denatured and digested with trypsin. Then, the variants of the carboxyterminal telopeptide of type I collagen were separated by high performance liquid chromatography (HPLC). The major histidinohydroxylysinonorleucine (HHL)-cross-linked variant was quantified by the SP4 assay, and the minor pyridinoline analogue (PA)-cross-linked telopeptide was quantified by the ICTP assay.nnnRESULTSnBoth the synthesis and degradation of type I collagen were increased (r=0.906; P<0.001) on the irradiated side, whereas the concentration of the MMP-2/TIMP-2 complex was decreased. In the insoluble tissue digests, the HHL-cross-linked telopeptides of type I collagen, also, when expressed/tissue hydroxyproline, were increased in the irradiated skin. TIMP-1, TIMP-2 or PA-cross-linked telopeptides of type I collagen showed no differences between the two sides.nnnCONCLUSIONSnRadiotherapy induces a long-term increase in the turnover of type I collagen and leads to the accumulation of cross-linked type I collagen in skin.

The production of collagen and the activity of mast-cell chymase increase in human skin after irradiation therapy

Abstract:u2002 Fibrosis is a common complication of radiotherapy. The pathogenesis of radiation‐induced fibrosis is not known in detail. There is increasing evidence to suggest that mast cells contribute to various fibrotic conditions. Several mast‐cell mediators have been proposed to have a role in fibrogenesis. Tryptase and chymase, the predominant proteins in mast cells, have been shown to induce fibroblast proliferation and collagen synthesis in vitro. In order to explore the role of mast cells in irradiation‐induced fibrosis, we analyzed skin biopsies and suction blister fluid (SBF) samples from the lesional and healthy‐looking skin of 10 patients who had been treated for breast cancer with surgery and radiotherapy. The biopsies were analyzed histochemically for mast‐cell tryptase, chymase, kit receptor, and tumor necrosis factor‐α. Skin collagen synthesis was assessed by determining the levels of type I and III procollagen amino‐terminal propeptides (PINP and PIIINP) in SBF and using immunohistochemical staining for PINP. Immunohistochemical stainings for prolyl‐4‐hydroxylase reflecting collagen synthesis and chymase immunoreactivity in irradiated and control skin were also performed. The mean level of procollagen propeptides in SBF, which reflects actual skin collagen synthesis in vivo, was markedly increased in irradiated skin compared to corresponding healthy control skin areas. The mean number of PINP‐positive fibroblasts was also significantly increased in the upper dermis of radiotherapy‐treated skin. The number of cells positive for tryptase, chymase and kit receptor was markedly increased in irradiated skin. In addition, using double‐staining techniques, it was possible to demonstrate that in some areas of the dermis, tryptase‐positive mast cells and fibroblasts are closely associated. These findings suggest a possible role of mast cells in enhanced skin collagen synthesis and fibrosis induced by radiotherapy.

Covalent binding of acetaldehyde to type III collagen

Incubation of neutral salt soluble type III pN-collagen with [14C]acetaldehyde in vitro resulted in the formation of spontaneously stable acetaldehyde-protein adducts. This reaction occurred primarily at lysine residues and it was not affected by 0.2-2 mM concentrations of ascorbate but addition of sodiumcyanoborohydride increased the stable adducts by 3-5-fold. When confluent cultures of human skin fibroblasts were incubated with physiologically relevant concentrations of acetaldehyde, it became covalently bound to type III procollagen secreted into the medium. We propose that acetaldehyde binding to collagen fibrils occurs in vivo following chronic alcohol consumption.

Radiation therapy induces tenascin expression and angiogenesis in human skin.

In analysing radiation-induced connective tissue changes, we studied tenascin expression, elastic fibres, angiogenesis and physio-mechanical properties in irradiated and contralateral healthy skin of radiotherapy-treated breast cancer patients. Skin biopsies were obtained from a radiotherapy-treated skin area and a corresponding non-treated skin area. Haematoxylin-eosin and Verhoeff stainings as well as immunohistochemical stainings for tenascin and factor VIII were performed. Epidermal and total skin thickness, together with the amount of elastic tissue calculated by computerized digital image analysis, were measured. Suction blisters were induced on both skin areas. Transepidermal water loss was analysed. Skin elasticity was also measured. Tenascin expression was found to be increased in irradiated human skin. In haematoxylin-eosin and factor VIlI-stained sections, an increase in the number of blood vessels was detected. Although skin stiffness measured by an elastometer was increased in irradiated skin, no marked difference in the elastic fibres could be found between treated and non-treated skin. The increased tenascin expression could be due to activation of cytokines as a result of irradiation. An increase in angiogenesis could be caused by an activation of angiogenetic factors by irradiation or due to direct radiation damage on blood vessel walls. Our findings suggest that the effects of irradiation tend to accumulate in the dermal parts of skin. The higher skin stiffness values measured by elastometer in irradiated skin could be due to an accumulation of dermal connective tissue as a result of irradiation.

Chlorate inhibits tyrosine sulfation of human type III procollagen without affecting its secretion or processing

Sodium chlorate, a potent inhibitor of sulfation reactions, completely inhibits the formation of tyrosine-o-sulfate in type III procollagen in human fibroblasts, when used in concentrations that do not affect the incorporation of radioactive amino acids into protein. The unsulfated type III procollagen is secreted into the medium at a rate comparable, to those of sulfated type III procollagen and type I procollagen, which normally does not undergo sulfation. The enzymatic cleavage of the aminoterminal propeptide of type III procollagen is incomplete in fibroblast cultures, irrespective of the sulfation status of the protein.