Clark M. Whitehead

Earlier studies of invasive breast tumors have shown that 60-80% are aneuploid and approximately 80% exhibit amplified centrosomes. In this study, we investigated the relationship of centrosome amplification with aneuploidy, chromosomal instability, p53 mutation, and loss of differentiation in human breast tumors. Twenty invasive breast tumors and seven normal breast tissues were analyzed by fluorescence in situ hybridization with centromeric probes to chromosomes 3, 7, and 17. We analyzed these tumors for both aneuploidy and unstable karyotypes as determined by chromosomal instability. The results were then tested for correlation with three measures of centrosome amplification: centrosome size, centrosome number, and centrosome microtubule nucleation capacity. Centrosome size and centrosome number both showed a positive, significant, linear correlation with aneuploidy and chromosomal instability. Microtubule nucleation capacity showed no such correlation, but did correlate significantly with loss of tissue differentiation. Centrosome amplification was detected in in situ ductal carcinomas, suggesting that centrosome amplification is an early event in these lesions. Centrosome amplification and chromosomal instability occurred independently of p53 mutation, whereas p53 mutation was associated with a significant increase in centrosome microtubule nucleation capacity. Together, these results demonstrate that independent aspects of centrosome amplification correlate with chromosomal instability and loss of tissue differentiation and may be involved in tumor development and progression. These results further suggest that aspects of centrosome amplification may have clinical diagnostic and/or prognostic value and that the centrosome may be a potential target for cancer therapy.

Exisulind (Aptosyn) is a novel antineoplastic drug being developed for the prevention and treatment of precancerous and malignant diseases. In colon tumor cells, the drug induces apoptosis by a mechanism involving cyclic GMP (cGMP) phosphodiesterase inhibition, sustained elevation of cGMP, and protein kinase G activation. We studied the effect of exisulind on bladder tumorigenesis induced in rats by the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine. Exisulind at doses of 800, 1000, and 1200 mg/kg (diet) inhibited tumor multiplicity by 36, 47, and 64% and tumor incidence by 31, 38, and 61%, respectively. Experiments on the human bladder tumor cell line, HT1376, showed that exisulind inhibited growth with a GI(50) of 118 microM, suggesting that the antineoplastic activity of the drug in vivo involved a direct effect on neoplastic urothelium. Exisulind also induced apoptosis as determined by DNA fragmentation, caspase activation, and morphology. Analysis of phosphodiesterase (PDE) isozymes in HT1376 cells showed PDE5 and PDE4 isozymes that were inhibited by exisulind with IC(50)s of 112 and 116 microM, respectively. Inhibition of PDE5 appears to be pharmacologically relevant, because treatment of HT1376 cells increased cGMP and activated protein kinase G at doses that induce apoptosis, whereas cyclic AMP levels were not changed. Immunocytochemistry showed that PDE5 was localized in discrete perinuclear foci in HT1376 cells. Immunohistochemistry showed that PDE5 was overexpressed in human squamous and transitional cell carcinomas compared with normal urothelium. The data lead us to conclude that future clinical trials of exisulind for human bladder cancer treatment and/or prevention should be considered and suggest a mechanism of action involving cGMP-mediated apoptosis induction.

We reported previously a significant increase in survival of nude rats harboring orthotopic A549 human non-small cell lung cancer tumors after treatment with a combination of exisulind (Sulindac Sulfone) and docetaxel (D. C. Chan, Clin. Cancer Res., 8: 904-912, 2002). The purpose of the current study was to determine the biochemical mechanisms responsible for the increased survival by an analysis of the effects of both drugs on A549 orthotopic lung tumors and A549 cells in culture. Orthotopic A549 rat lung tissue sections from drug-treated rats and A549 cell culture responses to exisulind and docetaxel were compared using multiple apoptosis and proliferation analyses [i.e., terminal deoxynucleotidyl transferase-mediated nick end labeling, active caspase 3, the caspase cleavage products cytokeratin 18 and p85 poly(ADP-ribose) polymerase, and Ki-67]. Immunohistochemistry was used to determine cyclic GMP (cGMP) phosphodiesterase (PDE) expression in tumors. The cGMP PDE composition of cultured A549 cells was resolved by DEAE-Trisacryl M chromatography and the pharmacological sensitivity to exisulind, and additional known PDE inhibitors were determined by enzyme activity assays. Exisulind inhibited A549 cell cGMP hydrolysis and induced apoptosis of A549 cells grown in culture. PDE5 and 1 cGMP PDE gene family isoforms identified in cultured cells were highly expressed in orthotopic tumors. The in vivo apoptosis rates within the orthotopic tumors increased 7-8-fold in animals treated with the combination of exisulind and docetaxel. Exisulind increased the in vivo apoptosis rates as a single agent. Docetaxel, but not exisulind, decreased proliferative rates within the tumors. The data indicate that exisulind-induced apoptosis contributed significantly to the increased survival in rats treated with exisulind/docetaxel. The mechanism of exisulind-induced apoptosis involves inhibition of cGMP PDEs, and these results are consistent with a cGMP-regulated apoptosis pathway.