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SUMMARY:Synovial sarcomas (SS) are characterized by a chromosomal translocation t(X;18)(p11.2;q11.2) which usually fuses the SYT gene from chromosome 18 to SSX1 or SSX2 genes on chromosome X. Also, a variant SYT-SSX4 fusion gene has recently been shown in a single SS case. In addition to these cytogenetic changes, bcl-2 expression, as assessed by immunohistochemistry, has been reported to be an almost general constitutive alteration of SS. In the present work, we analyze a series of 36 SS surgical samples (from 34 patients) by RT-PCR for the presence of the SYT-SSX1 or the SYT-SSX2 fusion transcript. The analysis was extended to SYT-SSX4 on SYT-SSX1-negative and SYT-SSX2-negative cases only. Our results showed a significant correlation between the SYT-SSX2 fusion and the monophasic SS histologic subtype. SYT-SSX1 fusion transcripts were present in both monophasic and biphasic tumors. The SYT-SSX4 fusion type was detected in a single monophasic SS. In the same series of SS cases, we also confirmed and extended the previously reported constitutive expression of bcl-2 protein, by using both immunohistochemical and western blot analysis. Moreover, we demonstrated that the BCL-2 gene is not rearranged or amplified at genomic level, indicating that the high levels of bcl-2 expression observed in SS might result from transcriptional activation of the gene and/or protein stabilization. Finally, we show that bcl-2 is not phosphorylated in tumors from patients who had been preoperatively treated with radio/chemotherapy, in tumors from untreated patients, or in an SS cell line (CME-1) after in vitro treatment with cytotoxic concentrations of DNA-damaging agents or taxanes. These data indicate that SS cells are unable to activate an apoptosis pathway involving bcl-2 phosphorylation/inactivation and may provide a possible explanation for the limited effectiveness of conventional pharmacological treatments of this tumor type. (Lab Invest 2000, 80:805-813).

The role of the site selectivity of topoisomerase II poisoning in the cytotoxic activity of anthracyclines has not been established. In this article, we have thus studied the levels and persistence of double-stranded DNA breaks (DSB) along with the cytotoxic activity in human leukemic HL60 cells of seven anthracyclines, including doxorubicin, daunorubicin, and idarubicin, as well as sugar-modified analogues characterized by an altered sequence specificity. Epimerization at the 3' position of the sugar moiety markedly affected the biological activity; indeed, a dramatic reduction of drug effects was evident for 3'-deamino-3'-epi-hydroxy-4'-deoxy-4'-amino-daunorubicin. The studied analogues could be gathered into three groups based on the DSB/cytotoxicity ratio. At equitoxic concentrations: (a) parent drugs and 3'-deamino-3'-epi-hydroxy-4'-deoxy-4'-amino-daunorubicin endowed with the same sequence specificity stimulated low DSB levels; (b) 3'-epi-daunorubicin and 3'-deamino-4'-deoxy-4'-epi-amino-idarubicin, which have a different sequence specificity, and teniposide (a structurally unrelated poison) stimulated higher amounts of DSB; and (c) 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin stimulated the highest DSB levels. For the last agent, a faster rate of cleavage resealing, which is consistent with a reduced DNA binding affinity, could account for the increased DSB/cytotoxicity ratio compared with parent drugs. However, for other analogues, the observed differences in DSB persistence/resealing could not completely explain the different DSB/cytotoxicity ratios. The results thus suggest that the cytotoxic potency of anthracyclines may be the result of an interplay of the level, the persistence, and the genomic localization of topoisomerase II-mediated DNA cleavage.

BBR3464 is a trinuclear platinum complex that exhibits a potent cytotoxicity and efficacy against cisplatin-resistant tumors. To better understand the determinants of cellular resistance to BBR3464, we selected a resistant ovarian carcinoma cell line after exposure to the complex. The resistant cells (A2780/BBR3464) exhibited a high level of resistance to the selecting agent, but a marginal cross-resistance to cisplatin. Although cellular accumulation of BBR3464 was similar in parental and in resistant cells, DNA platination was decreased in A2780/BBR3464 cells, suggesting a reduced drug accessibility to DNA. This behavior reflected a partial drug inactivation at cytoplasmic level, as a consequence of increased levels of nucleophilic molecules including metallothioneins and human neurofilament low, but not glutathione. A2780/BBR3464 cells also exhibited a reduced susceptibility to apoptosis, which was consistent with reduced expression of Bax, and an alteration of DNA mismatch repair system, as reflected by lack of expression of MLH1 and PMS2, which could impair the recognition/repair of DNA lesions. Whereas both platinum drugs induced G2/M arrest in the parental cells, BBR3464, but not cisplatin, caused a late G1 arrest of resistant cells. Cisplatin induced an appreciable increase of p21 WAF1 levels in both models, in contrast to BBR3464 that produced a substantial upregulation of p21 WAF1 only in parental cells. An inverse relationship with p21 WAF1 modulation was found for CHK1 in parental cells treated with both agents and in resistant cells treated with cisplatin. This pattern of response is consistent with a regulatory loop involving p53 and p21 WAF1 at G2 checkpoint. In contrast, no modulation of CHK1 was found in A2780/BBR3464 treated with the triplatinum compound. These findings, indicating a different activation of regulatory pathways at DNA damage checkpoints in response to cisplatin and BBR3464, support an altered ability of resistant cells to recognize or tolerate sublethal lesions induced by BBR3464. © 2003 Wiley-Liss, Inc. Key words: ovarian carcinoma; platinum drugs; resistancePlatinum compounds are antitumor agents effective in the treatment of human solid tumors, including ovarian carcinomas. 1 Development of resistance, however, is a major limitation of the efficacy of these compounds. [2][3][4] Over the past years, many platinum-containing drugs have been developed. Among these, mononuclear platinum complexes have shown molecular mechanisms similar to those of cisplatin. 5 The development of multinuclear platinum complexes has led recently to the identification of the trinuclear complex BBR3464. 6 -9 An investigation of the antitumor effects of BBR3464 in human tumor xenografts resistant or poorly responsive to cisplatin indicated a higher efficacy than cisplatin and a marked efficacy in p53 mutant carcinoma models. 8 Gene transfer studies documented a relevant role for p53 in regulating sensitivity to BBR3464. In particular, introduction of functional p53 in the SAOS osteosarcoma cell line n...

The heparan sulfate (HS) mimic/heparanase inhibitor roneparstat (SST0001) shows antitumor activity in preclinical sarcoma models. We hypothesized that this 100% N-acetylated and glycol-split heparin could interfere with the functions of several receptor tyrosine kinases (RTK) coexpressed in sarcomas and activated by heparin-binding growth factors. Using a phospho-proteomic approach, we investigated the drug effects on RTK activation in human cell lines representative of different sarcoma subtypes. Inhibition of FGF, IGF, ERBB and PDGF receptors by the drug was biochemically and functionally validated. Roneparstat counteracted the autocrine loop induced by the COL1A1/PDGFB fusion oncogene, expressed in a human dermatofibrosarcoma protuberans primary culture and in NIH3T3COL1A1/PDGFB transfectants, inhibiting cell anchorage-independent growth and invasion. In addition, roneparstat inhibited the activation of cell surface PDGFR and PDGFR-associated FAK, likely contributing to the reversion of NIH3T3COL1A1/PDGFB cell transformed and pro-invasive phenotype. Biochemical and histological/immunohistochemical ex vivo analyses confirmed a reduced activation of ERBB4, EGFR, INSR, IGF1R, associated with apoptosis induction and angiogenesis inhibition in a drug-treated Ewing's sarcoma family tumor xenograft. The combination of roneparstat with irinotecan significantly improved the antitumor effect against A204 rhabdoid xenografts resulting in a high rate of complete responses and cures. These findings reveal that roneparstat exerts a multi-target inhibition of RTKs relevant in the pathobiology of different sarcoma subtypes. These effects, likely cooperating with heparanase inhibition, contribute to the antitumor efficacy of the drug. The study supports heparanase/HS axis targeting as a valuable approach in combination therapies of different sarcoma subtypes providing a preclinical rationale for clinical investigation.