Bax deficiency extends the survival of Ku70 knockout mice that develop lung and heart diseases.

Ku70 (Lupus Ku autoantigen p70) is essential in nonhomologous end joining DNA double-strand break repair, and ku70(-/-) mice age prematurely because of increased genomic instability and DNA damage responses. Previously, we found that Ku70 also inhibits Bax, a key mediator of apoptosis. We hypothesized that Bax-mediated apoptosis would be enhanced in the absence of Ku70 and contribute to premature death observed in ku70(-/-) mice. Here, we show that ku70(-/-) bax(+/-) and ku70(-/-) bax(-/-) mice have better survival, especially in females, than ku70(-/-) mice, even though Bax deficiency did not decrease the incidence of lymphoma observed in a Ku70-null background. Moreover, we found that ku70(-/-) mice develop lung diseases, like emphysema and pulmonary arterial (PA) occlusion, by 3 months of age. These lung abnormalities can trigger secondary health problems such as heart failure that may account for the poor survival of ku70(-/-) mice. Importantly, Bax deficiency appeared to delay the development of emphysema. This study suggests that enhanced Bax activity exacerbates the negative impact of Ku70 deletion. Furthermore, the underlying mechanisms of emphysema and pulmonary hypertension due to PA occlusion are not well understood, and therefore ku70(-/-) and Bax-deficient ku70(-/-) mice may be useful models to study these diseases.

Optical pacing of the adult rabbit heart.

Optical pacing has been demonstrated to be a viable alternative to electrical pacing in embryonic hearts. In this study, the feasibility of optically pacing an adult rabbit heart was explored. Hearts from adult New Zealand White rabbits (n = 9) were excised, cannulated and perfused on a modified Langendorff apparatus. Pulsed laser light (λ = 1851 nm) was directed to either the left or right atrium through a multimode optical fiber. An ECG signal from the left ventricle and a trigger pulse from the laser were recorded simultaneously to determine when capture was achieved. Successful optical pacing was demonstrated by obtaining pacing capture, stopping, then recapturing as well as by varying the pacing frequency. Stimulation thresholds measured at various pulse durations suggested that longer pulses (8 ms) had a lower energy capture threshold. To determine whether optical pacing caused damage, two hearts were perfused with 30 µM of propidium iodide and analyzed histologically. A small number of cells near the stimulation site had compromised cell membranes, which probably limited the time duration over which pacing was maintained. Here, short-term optical pacing (few minutes duration) is demonstrated in the adult rabbit heart for the first time. Future studies will be directed to optimize optical pacing parameters to decrease stimulation thresholds and may enable longer-term pacing.

Inhibition of metastasis by HEXIM1 through effects on cell invasion and angiogenesis.

We report on the role of hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) as an inhibitor of metastasis. HEXIM1 expression is decreased in human metastatic breast cancers when compared with matched primary breast tumors. Similarly we observed decreased expression of HEXIM1 in lung metastasis when compared with primary mammary tumors in a mouse model of metastatic breast cancer, the polyoma middle T antigen (PyMT) transgenic mouse. Re-expression of HEXIM1 (through transgene expression or localized delivery of a small molecule inducer of HEXIM1 expression, hexamethylene-bis-acetamide) in PyMT mice resulted in inhibition of metastasis to the lung. Our present studies indicate that HEXIM1 downregulation of HIF(-)1α protein allows not only for inhibition of vascular endothelial growth factor-regulated angiogenesis, but also for inhibition of compensatory pro-angiogenic pathways and recruitment of bone marrow-derived cells (BMDCs). Another novel finding is that HEXIM1 inhibits cell migration and invasion that can be partly attributed to decreased membrane localization of the 67 kDa laminin receptor, 67LR, and inhibition of the functional interaction of 67LR with laminin. Thus, HEXIM1 re-expression in breast cancer has therapeutic advantages by simultaneously targeting more than one pathway involved in angiogenesis and metastasis. Our results also support the potential for HEXIM1 to indirectly act on multiple cell types to suppress metastatic cancer.

HEXIM1 modulates vascular endothelial growth factor expression and function in breast epithelial cells and mammary gland.

Recently, we found that mutation of the C-terminus of transcription factor hexamethylene bisacetamide-inducible protein 1 (HEXIM1) in mice leads to abnormalities in cardiovascular development because of aberrant vascular endothelial growth factor (VEGF) expression. HEXIM1 regulation of some genes has also been shown to be positive transcription elongation factor b (P-TEFb) dependent. However, it is not known whether HEXIM1 regulates VEGF in the mammary gland. We demonstrate that HEXIM1 regulates estrogen-induced VEGF transcription through inhibition of estrogen receptor-alpha recruitment to the VEGF promoter in a P-TEFb-independent manner in MCF-7 cells. Under hypoxic conditions, HEXIM1 inhibits estrogen-induced hypoxia-inducible factor-1 alpha (HIF-1alpha) protein expression and recruitment of HIF-1alpha to the hypoxia-response element in the VEGF promoter. In the mouse mammary gland, increased HEXIM1 expression decreased estrogen-driven VEGF and HIF-1alpha expression. Conversely, a mutation in the C-terminus of HEXIM1 (HEXIM1(1-312)) led to increased VEGF and HIF-1alpha expression and vascularization in mammary glands of heterozygous HEXIM1(1-312) mice when compared with their wild-type littermates. In addition, HEXIM1(1-312) mice have a higher incidence of carcinogen-induced mammary tumors with increased vascularization, suggesting an inhibitory role for HEXIM1 during angiogenesis. Taken together, our data provide evidence to suggest a novel role for HEXIM1 in cancer progression.