Mechanism of anthracycline-mediated down-regulation of GATA4 in the heart.

AIMS: Anthracyclines such as daunorubicin (DNR) and doxorubicin are effective cancer chemotherapeutic agents, but can induce cardiotoxicity. GATA4 has been shown to serve as a survival factor of cardiac muscle cells, and anthracyclines promote apoptosis in part by down-regulating GATA4. The present study investigated the mechanism of anthracycline action to down-regulate GATA4. METHODS AND RESULTS: DNR inhibited the transcriptional activity exhibited by the 250 bp conserved region immediately upstream from the transcriptional start site of the Gata4 gene. Mapping this region identified that the CCAAT-binding factor/nuclear factor-Y (CBF/NF-Y) binding to the CCAAT box was inhibited by DNR in HL-1 cardiac muscle cells and in perfused isolated mouse hearts. The DNR action on the Gata4 promoter was found to be dependent on p53, since DNR promoted nuclear binding of p53 to CBF/NF-Y and pifithrin-α (a p53 inhibitor) attenuated DNR down-regulation of GATA4. CONCLUSION: Anthracycline down-regulation of GATA4 is mediated by the inhibition of Gata4 gene transcription via a novel mechanism that involves the p53-dependent inhibition of CBF/NF-Y binding to the CCAAT box within the Gata4 promoter.

Acute intermittent hypoxia activates myocardial cell survival signaling.

Intermittent hypoxia (IH) with repeated episodes of hypoxia-normoxia cycle has been shown to exert preconditioning-like cardioprotective effects. To understand the mechanism of these events, we investigated the changes in cardiac gene expression in response to acute IH. Mice were subjected to five cycles of 2 min of 10% O(2) plus 2 min of 21% O(2). RNA was isolated, and gene array analysis was performed. Results show that the expression of antiapoptotic genes, such as Bcl-2 and Bcl-x(L), were increased after acute IH. GATA-4 regulates transcription of these genes, and, consistently, GATA-4 activity was increased by acute IH. Although the phosphorylation of GATA-4 has been shown to regulate its activity, no changes in GATA-4 phosphorylation status by acute IH were noted. Gene transcription of gata4 was increased by acute IH, and this might be responsible for the enhanced GATA activity. To understand the mechanism of acute IH activation of gata4 gene transcription, we identified a promoter region of the mouse gata4 gene that is 1,000 bp immediately upstream from the transcriptional start site. In cardiac muscle cells, truncation of 1,000 to 250 bp did not alter the transcriptional activity, suggesting that the proximal 250-bp region contains important transcriptional regulatory sites. We further found that acute IH activates factors which bind to the proximal 100-bp region. Thus acute IH activates not yet identified factors that bind to the proximal 100-bp region of the gata4 promoter and, in turn, increases gata4 gene transcription, leading to enhanced expression of Bcl-2 and Bcl-x(L).

Hematopoietic stem cells mobilization and immune response in tumor-bearing mice.

INTRODUCTION: Malignant diseases are known to modulate the number and function of myeloid, erythroid, and lymphoid cells. Since these cells are derived from hematopoietic stem cells (HSC), it is not clear if the observed effects of cancer on such cells are direct or indirect via stem cells. The purpose of this study was to evaluate the effect of breast cancer upon the levels and activity of peripheral blood hematopoietic stem cells. MATERIALS AND METHODS: Four weeks following the establishment of 4T1 breast cancers in BALB/c mice, the animals were killed, blood and spleen harvested, and processed for light density mononuclear cells. Colony forming unit in culture assay was used to determine the activity of HSCs. Flow cytometry was used to determine the levels of lineage negative HSCs expressing c-kit and Sca-1 antigen (Lin c-kitSca-1). Mitogenic, cytotoxic and ELISPOT assays were used to evaluate functional properties of cells. Plasma cytokine levels were determined with ELISA assay. RESULTS: In tumor-bearing mice, there was a 2- and 4-fold increase in the levels and proliferative capacity of HSCs, respectively, compared with controls. Contemporaneously, there was a 13-fold increase in plasma G-CSF in tumor-bearing animals compared with controls (0.225 ng/ml versus 3.0 ng/ml). Furthermore, the number of interferon gamma-secreting cells was significantly increased in tumor-bearing animals. Concurrently, cytotoxic activity of NK cells was significantly increased in tumor-bearing animals as compared with controls (22.4 +/- 10.6 versus 10.3 +/- 2.95; P < 0.05). SUMMARY: These results suggest that (1) breast cancer mobilizes hematopoietic stem cells in mice presumably through G-CSF production, and (2) that such cancer-mobilized stem cells give rise to immune cell lineages which are functionally hyperactive in their cytotoxic activities. Such cells could be expected to have appreciable therapeutic benefit in terms of cancer cell cytotoxic activity when used as part of stem cell transplantation therapy in cancer patients.