Doxorubicin induces cell senescence preferentially over apoptosis in the FU-SY-1 synovial sarcoma cell line.

Surgical resection coupled with adjuvant radiotherapy and/or doxorubicin based chemotherapy are the mainstays of synovial sarcoma (SS) treatment. Although effective as a SS adjuvant, the proposed mechanism of action of doxorubicin remains controversial. Current opinion supports DNA damage-induced apoptosis. This in vitro study used cDNA gene expression profiling to investigate whether apoptosis, alone or in combination with cell senescence, is induced by doxorubicin in SS cells. Cell cultures of the FU-SY-1 SS, the pleomorphic SW982 sarcoma, and a primary dermal fibroblast (NHDF), were exposed to 500 nM doxorubicin, and then processed for cDNA microarray analysis. The one class response option of SAM (Significance Analysis of Microarrays) was used to test for significant overexpression of 15 apoptosis-related genes and nine senescence-related genes. Drug-induced cell senescence was quantified by measuring beta-galactosidase activity. None of 15 apoptosis-related genes and only two of nine senescence-related genes were identified by SAM as significantly overexpressed in doxorubicin-treated cultures. Drug-induced senescence as reflected by beta-galactosidase activity was significantly increased (p < 0.05) only in FU-SY-1 SS cultures. Apoptosis does not appear to be a major determinant of doxorubicin-induced mortality in FU-SY-1 SS or NHDF cultures, but may impact SW982 cells via the overexpression of BAX relative to Bcl-2. Doxorubicin-induced cell senescence was prominent in FU-SY-1 SS cultures, but negligible in SW982 and NHDF cultures. Likely, both apoptosis and cell senescence contribute to doxorubicin-induced cell death in this synovial sarcoma cell line.

G3139 antisense oligonucleotide directed against antiapoptotic Bcl-2 enhances doxorubicin cytotoxicity in the FU-SY-1 synovial sarcoma cell line.

Synovial sarcoma (SS) is a highly aggressive, periarticular soft tissue sarcoma that causes death in more than half of affected children, adolescents, and young adults. Five- and 10-year survival rates are as low as 36 and 20%, respectively. Bcl-2, a negative regulator of apoptosis, is overexpressed in up to 90% of SS. Increased Bcl-2 expression not only leads to the development of cancer, but also to resistance of many anticancer chemotherapeutic agents. We hypothesized reducing Bcl-2 expression in SS should enhance doxorubicin cytotoxicity. Cell cultures representing two human sarcomas (FU-SY-1 SS and the pleomorphic SW982) and a primary human dermal fibroblast comparator (NHDF) were exposed in vitro to doxorubicin, or to doxorubicin preceded by Bcl-2 (G3139) antisense oligonucleotides, and assayed for cell survival, apoptosis, and modulations in Bcl-2 and Bcl-xL mRNA and protein content. SW982 sarcoma cells proved most susceptible to doxorubicin, while NHDF mesenchymal cells were least sensitive to doxorubicin. Treatment of FU-SY-1 SS with G3139 reduced Bcl-2 mRNA and protein levels, which enhanced doxorubicin-induced cell killing. There was a concurrent reduction in Bcl-xL mRNA following G3139 application in FU-SY-1 and NHDF cultures, but not in SW982. G3139 anti-Bcl-2 intervention sensitized the FU-SY-1 SS to doxorubicin, due to increased apoptosis. G3139 intervention was ineffective in the two non-SS cell lines.

Transit tumor retrieval preserves RNA fidelity and obviates snap-freezing.

UNLABELLED: Genetic expression profiling is enabling investigators to discover new diagnostic and possibly therapeutic pathways in sarcoma biology. To draw substantial conclusions from these molecular analyses, adequate tissue samples must be accrued. Beyond cohort size, the most variable and limiting aspect of doing gene expression analyses on fresh human tissue is the preservation of labile ribonucleic acids extracted from clinical specimens. We have developed a novel retrieval protocol that is readily amenable to the clinical constraints placed on surgeons and pathologists that minimizes variables that can corrupt ribonucleic acid fidelity. We evaluate critically genomic message integrity of mesenchymal tumors derived from transcontinental inter-institutional collaboration. Intact total ribonucleic acid was isolated and assessed for quality and quantity. Ribosomal RNA integrity was quantified using a bioanalyzer. Ribonucleic acid from 42 mesenchymal tumors was isolated and quantified, with selected samples amplified. The mean ribosomal ratios for collaborative institutions ranged from 1.0 to 1.18. Samples remained at 4 degrees C before processing from 1 to 17 days. Tumors stabilized using this protocol retained total ribonucleic acid integrity suitable for amplification and genomic expression analysis regardless of the institutional source or preprocessing duration, enabling a potential consortium of investigators to collaborate in the expression profiling of sarcomas. LEVEL OF EVIDENCE: Diagnostic study, Level III-3 (no consistently applied gold standard). See the Guidelines for Authors for a complete description of levels of evidence.