Type V collagen regulates the expression of apoptotic and stress response genes by breast cancer cells.
Type V collagen is a "minor" component of normal human breast stroma, which is subjected to over-deposition in cases of ductal infiltrating carcinoma (DIC). We reported that, if used as a culture substrate for the DIC cell line 8701-BC, it exhibited poorly-adhesive properties and restrained the proliferative and motile behavior of the cell subpopulation able to attach onto it. Moreover, this collagen species was able to trigger DNA fragmentation and impair survival of 8701-BC cells. In this study, we have extended our investigation with the aim to obtain further evidence that the death induced by type V collagen was of the apoptotic type by (i) microscopic detection and quantitation of Apoptag-labeled cells, (ii) analysis of the expression levels of selected genes coding for apoptosis-linked factors, caspases, and stress-response proteins by conventional and semi-quantitative multiplex PCR, and (iii) evaluation of the extent of caspase activation by chromogenic assay. We report here that type V collagen is able to determine an increase in the percentage of Apoptag-positive cells, to up-regulate Bcl-xS, Bad, Dap kinase, hsf-1, mthsp75, caspase-1, -5, -8, -9, and -14, whilst down-regulating Bcl-2, Bcl-xbeta, and hsp60. Treatment of cell lysates with chromogenic tetrapeptide substrates specific for caspase-1, -5, -8, and -9 demonstrated a marked increase of enzymatic activity in the presence of type V collagen. Our data validate 8701-BC cell line as a suitable "in vitro" model for further and more detailed studies on the molecular mechanisms of the death response induced by type V collagen on primary DIC cells.
Luparello
Claudio
C
Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, Viale delle Scienze, Palermo, Italy. clupar@tin.it
Sirchia
Rosalia
R
D017209:Apoptosis / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D018270:Carcinoma, Ductal, Breast / Q000235:genetics* / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D024062:Collagen Type V / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans
D024062:Collagen Type V; D006360:Heat-Shock Proteins; D020169:Caspases