Direct optic nerve sheath (DONS) application of Schwann cells prolongs retinal ganglion cell survival in vivo.

Cell-based therapies are increasingly recognized as a potential strategy to treat retinal neurodegenerative disease. Their administration, however, is normally indirect and complex, often with an inability to assess in real time their effects on cell death and their migration/integration into the host retina. In the present study, using a partial optic nerve transection (pONT) rat model, we describe a new method of Schwann cell (SC) delivery (direct application to injured optic nerve sheath, SC/DONS), which was compared with intravitreal SC delivery (SC/IVT). Both SC/DONS and SC/IVT were able to be assessed in vivo using imaging to visualize retinal ganglion cell (RGC) apoptosis and SC retinal integration. RGC death in the pONT model was best fitted to the one-phase exponential decay model. Although both SC/DONS and SC/IVT altered the temporal course of RGC degeneration in pONT, SC/DONS resulted in delayed but long-lasting effects on RGC protection, compared with SC/IVT treatment. In addition, their effects on primary and secondary degeneration, and axonal regeneration, were also investigated, by histology, whole retinal counting, and modelling of RGC loss. SC/DONS was found to significantly reduce RGC apoptosis in vivo and significantly increase RGC survival by targeting secondary rather than primary degeneration. Both SC/DONS and SC/IVT were found to promote RGC axonal regrowth after optic nerve injury, with evidence of GAP-43 expression in RGC somas and axons. SC/DONS may have the potential in the treatment of optic neuropathies, such as glaucoma. We show that SC transplantation can be monitored in real time and that the protective effects of SCs are associated with targeting secondary degeneration, with implications for translating cell-based therapies to the clinic.

Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.

Mammalian cells can respond to damage or stress by entering a state of arrested growth and altered function termed cellular senescence. Several lines of evidence suggest that the senescence response suppresses tumorigenesis. Cellular senescence is also thought to contribute to aging, but the mechanism is not well understood. We show that senescent human fibroblasts stimulate premalignant and malignant, but not normal, epithelial cells to proliferate in culture and form tumors in mice. In culture, the growth stimulation was evident when senescent cells comprised only 10% of the fibroblast population and was equally robust whether senescence was induced by replicative exhaustion, oncogenic RAS, p14(ARF), or hydrogen peroxide. Moreover, it was due at least in part to soluble and insoluble factors secreted by senescent cells. In mice, senescent, much more than presenescent, fibroblasts caused premalignant and malignant epithelial cells to form tumors. Our findings suggest that, although cellular senescence suppresses tumorigenesis early in life, it may promote cancer in aged organisms, suggesting it is an example of evolutionary antagonistic pleiotropy.

Id-1, ITF-2, and Id-2 comprise a network of helix-loop-helix proteins that regulate mammary epithelial cell proliferation, differentiation, and apoptosis.

Mammary epithelial cells proliferate, invade the stroma, differentiate, and die in adult mammals by mechanisms that are poorly understood. We found that Id-1, an inhibitor of basic helix-loop-helix transcription factors, regulates mammary epithelial cell growth, differentiation, and invasion in culture. Here, we show that Id-1 is expressed highly during mammary development in virgin mice and during early pregnancy, when proliferation and invasion are high. During mid-pregnancy, Id-1 expression declined to undetectable levels as the epithelium differentiated fully. Surprisingly, Id-1 increased during involution, when the epithelium undergoes extensive apoptosis. To determine whether Id-1 regulates both proliferation and apoptosis, we constitutively expressed Id-1 in mammary epithelial cell cultures. Id-1 stimulated proliferation in sparse cultures but induced apoptosis in dense cultures, which reflect epithelial cell density during early pregnancy and involution, respectively. To understand how Id-1 acts, we screened a yeast two-hybrid library from differentiating mammary epithelial cells and identified ITF-2, a basic helix-loop-helix transcription factor, as an Id-1-interacting protein. Overexpression of ITF-2 significantly reduced Id-1-stimulated proliferation and apoptosis. We show further that, in contrast to Id-1, Id-2 was expressed highly in differentiated mammary epithelial cells in vivo and in culture. In culture, Id-2 antisense transcripts blocked differentiation. Our results suggest that Id-1, ITF-2, and Id-2 comprise a network of interacting molecular switches that govern mammary epithelial cell phenotypes.

Molecular cloning and characterization of a zinc finger protein involved in Id-1-stimulated mammary epithelial cell growth.

Id proteins are dominant negative regulators of basic helix-loop-helix transcription factors. Previous work in our laboratory has shown that constitutive expression of Id-1 in SCp2 mouse mammary epithelial cells inhibits their differentiation and induces proliferation, invasion, and migration. Id-1 expression also correlates with the invasive and aggressive potential of human breast cancer cells. However, little is known about Id-1 target genes that are important for regulating normal and transformed breast epithelial cell phenotypes. Now we report the cloning of a novel zinc finger protein, Zfp289, using degenerate primers to specifically amplify cDNAs from Id-1-transfected SCp2 cells. Zfp289 has homology with a yeast zinc finger protein, the GTPase-activating protein Gcs-1, which was initially identified as a gene required for the re-entry of cells into the cell cycle after stationary phase growth. Zfp289 mRNA expression pattern correlates with Id-1 expression in SCp2 mammary epithelial cells under various experimental conditions as well as in the mouse mammary gland at different stages of development. It is predominantly present in the cytoplasm of the cells as evident from green fluorescent protein fusion protein localization. SCp2 mammary epithelial cells with constitutive expression of Zfp289 have a higher S-phase index, compared with control cells, when cultured in a serum-free medium. We conclude that the novel zinc finger protein Zfp289, which may represent the mammalian homologue of Gcs-1, is potentially an important mediator of the Id-1-induced proliferation pathway in mammary epithelial cells.

A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells.

The helix-loop-helix protein Id-1 inhibits the activity of basic helix-loop-helix transcription factors, and is an important regulator of cell growth and tissue-specific differentiation. We have shown (P. Y. Desprez et al., Mol. Cell. Biol., 18: 4577-4588, 1998) that ectopic expression of Id-1 inhibits differentiation and stimulates the proliferation and invasiveness of mouse mammary epithelial cells, and that there is a correlation between the levels of Id-1 protein and the aggressiveness of several human breast cancer cell lines. Here, we show that aggressive and metastatic breast cancer cells express high levels of Id-1 mRNA because of a loss of serum-dependent regulation that is mediated by a 2.2-kb region of the human Id-1 promoter. Three lines of evidence suggest that unregulated Id-1 expression may be an important regulator of the aggressive phenotype of a subset of human breast cancer cells: (a) a constitutively expressed Id-1 cDNA, when introduced into a nonaggressive breast cancer cell line (T47D), conferred a more aggressive phenotype, as measured by growth and invasiveness; (b) Id-1 was an important mediator of the effects of sex steroid hormones on T47D cell proliferation. Estrogen stimulated proliferation and induced Id-1 expression, whereas progesterone inhibited proliferation and repressed Id-1 expression. Progesterone repressed Id-1 expression, at least in part by repressing transcription. Most importantly, an antisense oligonucleotide that reduced Id-1 protein levels reduced the ability of estrogen to stimulate cell proliferation, whereas constitutive Id-1 expression rendered cells refractory to growth inhibition by progesterone; and (c) using a limited number of breast cancer biopsies, we showed that Id-1 was more frequently expressed in infiltrating carcinomas compared with ductal carcinomas in situ. Our results suggest that Id-1 can control the malignant progression of breast cancer cells, particularly that mediated by sex steroid hormones. Moreover, Id-1 has the potential to serve as a marker for aggressive breast tumors.

Regulation of mammary epithelial cell phenotypes by the helix-loop-helix protein, Id-1.

Mammary epithelial cells undergo cycles of proliferation, invasion, differentiation and apoptotic cell-death throughout adult life. The molecular mechanisms that regulate these complex and co-ordinated developmental programs are poorly understood. We have identified Id-1 protein, a negative regulator of basic helix-loop-helix transcription factors, as a critical regulator of these normal mammary epithelial cell phenotypes. We also found that Id-1 is an important regulator of the aggressive and invasive phenotype, as well as mediator of the effects of sex steroid hormones, in human breast cancer cells.