John T. Barrett

A role for macroautophagy in protection against 4-hydroxytamoxifen-induced cell death and the development of antiestrogen resistance

This study identifies macroautophagy as a key mechanism of cell survival in estrogen receptor–positive (ER+) breast cancer cells undergoing treatment with 4-hydroxytamoxifen (4-OHT). This selective ER modifier is an active metabolite of tamoxifen commonly used for the treatment of breast cancer. Our study provides the following key findings: (a) only 20% to 25% of breast cancer cells treated with 4-OHT in vitro die via caspase-dependent cell death; more typically, the antiestrogen-treated ER+ breast cancer cells express increased levels of macroautophagy and are viable; (b) 4-OHT–induced cell death, but not 4-OHT–induced macroautophagy, can be blocked by the pan-caspase inhibitor z-VAD-fmk, providing strong evidence that these two outcomes of antiestrogen treatment are not linked in an obligatory manner; (c) 4-OHT–resistant cells selected from ER+ breast cancer cells show an increased ability to undergo antiestrogen-induced macroautophagy without induction of caspase-dependent cell death; and (d) 4-OHT, when used in combination with inhibitors of autophagosome function, induces robust, caspase-dependent apoptosis of ER+, 4-OHT–resistant breast cancer cells. To our knowledge, these studies provide the first evidence that macroautophagy plays a critical role in the development of antiestrogen resistance. We propose that targeting autophagosome function will improve the efficacy of hormonal treatment of ER+ breast cancer. [Mol Cancer Ther 2008;7(9):2977–87]

Autophagy facilitates the progression of ERα-positive breast cancer cells to antiestrogen resistance

A major impediment to the successful treatment of estrogen receptor α (ERα)-positive breast cancer is the development of antiestrogen resistance. Tamoxifen, the most commonly used antiestrogen, exerts its pharmacological action by binding to ERα and blocking the growth- promoting action of estrogen-bound ERα in breast cancer cells. Tamoxifen treatment primarily induces cytostasis (growth arrest) and the surviving breast cancer cells commonly acquire tamoxifen resistance. Numerous clinically-relevant mechanisms of acquired antiestrogen resistance have been identified by in vitro studies. Our recent studies (Mol Cancer Ther 2008: 7:2977-87) now demonstrate that autophagy (also referred to as macroautophagy) is critical to the development of antiestrogen resistance. Under conditions of compromised autophagy, including treatments with pharmacological inhibitors and RNAi targeting of the beclin 1 gene, the cytotoxicity (death-inducing effects) of the antiestrogen 4-hydroxytamoxifen (4-OHT) was significantly increased. 4-OHT is an active metabolite of tamoxifen commonly used for in vitro studies. A step-wise drug selection protocol, using 4-OHT as the selecting drug, established antiestrogen-resistant breast cancer cell lines. Analysis of a representative resistant cell line showed an increased ability of the cells to sustain high levels of antiestrogen-induced autophagy without progression to death. Importantly, blockade of autophagosome function in the 4-OHT-treated, antiestrogen-resistant cells induced a robust death response. These data provide strong evidence that autophagy is a key mechanism of cell survival during antiestrogen challenge and progression to antiestrogen resistance. We discuss the potential benefit of blocking autophagosome function to significantly reduce the emergence of antiestrogen-resistant breast cancer cells.

Mifepristone induces growth arrest, caspase activation, and apoptosis of estrogen receptor-expressing, antiestrogen-resistant breast cancer cells

Purpose: A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study, we sought to determine whether antiprogestin treatment, used as a monotherapy or in combination with antiestrogen therapy, induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells. Experimental Design: MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant), and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays, cell counting, flow cytometry, and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase. Results: All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity, including resistance to physiological levels of 4-OHT. Importantly, all of the clones were sensitive to the antiprogestin MIF, whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation, G1 arrest, and apoptosis preceded by caspase activation. Conclusions: We demonstrate that: (a) estrogen receptor+progesterone receptor+, 4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and, ultimately, improve the therapeutic index of antiestrogen therapy.

Quantification of Ionizing Radiation-Induced Cell Death In Situ in a Vertebrate Embryo

Abstract Bladen, C. L., Flowers, M., Miyake, K., Podolsky, R. H., Barrett, J., Kozlowski, D. J. and Dynan, W. S. Quantification of Ionizing Radiation-Induced Cell Death In Situ in a Vertebrate Embryo. Radiat. Res. 168, 149–157 (2007). Quantitative studies of radiation cytotoxicity have been performed mostly in cells in culture. For a variety of reasons, however, the response of cells in culture may not reflect the response for cells in situ in a whole organism. We describe here an approach for quantification of radiation-induced cell death in vivo using the transparent embryo of the zebrafish, Danio rerio, as a model vertebrate system. Using this system, we show that the number of TUNEL-positive cells within a defined region increases approximately linearly with radiation dose up to 1 Gy. The results are consistent with predictions of a linear-quadratic model. The use of alternative models, accommodating a response threshold or low-dose hypersensitivity, did not significantly improve the fit to the observed data. Attenuation of the expression of the 80-kDa subunit of Ku, an essential protein for the nonhomologous end-joining pathway of repair, led to a dose reduction of 30- to 34-fold, possibly approaching the limit where each double-strand break causes a lethal hit. In both the Ku80-attenuated and the control embryos, apoptotic cells were distributed uniformly, consistent with a cell-autonomous mechanism of cell death. Together, these results illustrate the potential of the zebrafish for quantitative studies of radiation-induced cell death during embryogenesis and in vivo.

Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes

PURPOSE To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. MATERIALS AND METHODS The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed. RESULTS A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. CONCLUSIONS Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.

Intracranial Fibrosarcoma Arising 5 Years after Chemotherapy Alone for Glioblastoma multiforme in a Child

We present a child diagnosed with glioblastoma multiforme during infancy and successfully treated with the ‘eight-in-one’ chemotherapy regimen, who developed an intracranial fibrosarcoma 5 years later. After resection of the fibrosarcoma, she received cranial radiation therapy and high dose chemotherapy with bone marrow transplant. She remains alive and recurrence-free 7 years following the diagnosis of her second intracranial malignancy.

Ga-67 used to localize the tumor bed for radiation therapy after lumpectomy.

The authors present a new method to locate the tumor bed after lumpectomy. The method relies on accumulation of Ga-67 at the surgical site. This technique was useful in identifying the tumor bed in six candidates for breast conserving surgery and radiation therapy. This method may be applicable in other soft tissue malignancies that require postoperative radiation.