Breaking the 'harmony' of TNF-α signaling for cancer treatment.

Tumor necrosis factor-alpha (TNF-α) binds to two distinct receptors, TNFR1/p55 and TNFR2/p75. TNF-α is implicated in the processes of tumor growth, survival, differentiation, invasion, metastases, secretion of cytokines and pro-angiogenic factors. We have shown that TNFR2/p75 signaling promotes ischemia-induced angiogenesis via modulation of several angiogenic growth factors. We hypothesized that TNFR2/p75 may promote tumor growth and angiogenesis. Growth of mouse Lewis lung carcinoma (LLC1) and/or mouse melanoma B16 cell was evaluated in wild type (WT), p75 knockout (KO) and double p55KO/p75KO mouse tumor xenograft models. Compared with WT and p55KO/p75KO mice, growth of tumors in p75KO mice was significantly decreased (twofold) in both LLC and B16 tumors. Tumor growth inhibition was correlated with decreases in vascular endothelial growth factor (VEGF) expression and capillary density, as well as bone marrow-derived endothelial progenitor cells incorporation into the functional capillary network, and an increase in apoptotic cells in LLC xenografts. Gene array analysis of tumor tissues showed a decrease in gene expression in pathways that promote tumor angiogenesis and cell survival. Blocking p75 by short-hairpin RNA in cultured LLCs led to increases in TNF-mediated apoptosis, as well as decreases in the constitutive and TNF-mediated expression of angiogenic growth factors (VEGF, HGF, PLGF), and SDF-1α receptor CXCR4. In summary, p75 is essential for tumor angiogenesis and survival in highly vascularized murine lung tumor xenografts. Blocking p75 expression may lead to tumor regression. This may represent new and effective therapy against lung neoplasms and potentially tumors of other origin.

Ageing is associated with diminished apoptotic cell clearance in vivo.

Ageing leads to immune system dysfunction and the accumulation of autoantibodies. Because the rapid phagocytic clearance of apoptotic cells is required to prevent the development of autoimmunity, we examined the relative clearance of apoptotic material in young and aged mice using two independent assays. First, 2-year-old mice were found to be impaired in their ability to clear apoptotic keratinocytes following ultraviolet irradiation of the skin. Secondly, peritoneal macrophages exposed to apoptotic Jurkat T cells in vivo displayed diminished phagocytic activity in aged mice compared with 8-week-old mice. Consistent with these findings, aged mice exhibited signs of autoimmunity with the appearance of anti-nuclear antibodies and increased kidney glomerular size as well as complement deposits within the glomeruli. In vitro assays revealed that the pretreatment of macrophages with the serum from aged mice led to a reduction in their ability to phagocytose apoptotic bodies compared with macrophages treated with serum from young mice. These data show that the ageing process is accompanied by a diminished ability to clear apoptotic debris. This accumulation of apoptotic debris could contribute to immune system dysfunction that occurs in aged organisms.

Dietary lutein/zeaxanthin partially reduces photoaging and photocarcinogenesis in chronically UVB-irradiated Skh-1 hairless mice.

Lutein and zeaxanthin are xanthophyll carotenoids with potent antioxidant properties protecting the skin from acute photodamage. This study extended the investigation to chronic photodamage and photocarcinogenesis. Mice received either a lutein/zeaxanthin-supplemented diet or a standard nonsupplemented diet. Dorsal skin of female Skh-1 hairless mice was exposed to UVB radiation with a cumulative dose of 16,000 mJ/cm(2) for photoaging and 30,200 mJ/cm(2) for photocarcinogenesis. Clinical evaluations were performed weekly, and the animals were sacrificed 24 h after the last UVB exposure. For photoaging experiments, skin fold thickness, suprapapillary plate thickness, mast cell counts and dermal desmosine content were evaluated. For photocarcinogenesis, samples of tumors larger than 2 mm were analyzed for histological characterization, hyperproliferation index, tumor multiplicity, total tumor volume and tumor-free survival time. Results of the photoaging experiment revealed that skin fold thickness and number of infiltrating mast cells following UVB irradiation were significantly less in lutein/zeaxanthin-treated mice when compared to irradiated animals fed the standard diet. The results of the photocarcinogenesis experiment were increased tumor-free survival time, reduced tumor multiplicity and total tumor volume in lutein/zeaxanthin-treated mice in comparison with control irradiated animals fed the standard diet. These data demonstrate that dietary lutein/zeaxanthin supplementation protects the skin against UVB-induced photoaging and photocarcinogenesis.

Overexpression of p27(Kip1) by doxycycline-regulated adenoviral vectors inhibits endothelial cell proliferation and migration and impairs angiogenesis.

Formation of new blood vessels in the adult animal (i.e., angiogenesis) is an important event for tissue repair and for tumor growth and metastasis. Angiogenesis involves the migration and proliferation of endothelial cells. We have investigated the role of the growth suppressor p27(Kip1) (p27) on endothelial cell function in vitro and angiogenesis in vivo. We have generated Ad-TetON, a replication-deficient adenovirus that constitutively expresses the reverse tet-responsive transcriptional activator, and Ad-TRE-p27, which drives expression of p27 under the control of the tet response element. Western blot analysis demonstrated doxycycline-dependent overexpression of p27 in human umbilical vein endothelial cells (HUVECs) coinfected with Ad-TetON and Ad-TRE-p27, which resulted in a marked inhibition of DNA replication and cell migration in vitro. Inducible overexpression of p27 in cultured HUVECs inhibited the formation of tubelike structures and, when applied in a murine model of hind limb ischemia, reduced hind limb blood flow recovery and capillary density. These findings thus underscore a novel role of p27 in regulating endothelial cell migration in vitro and angiogenesis in vivo, suggesting a novel anti-angiogenic therapy based on inducible p27 overexpression.

Inhibition of the cyclin D1/E2F pathway by PCA-4230, a potent repressor of cellular proliferation.

1. Tight control of cellular growth is essential to ensure normal tissue patterning and prevent pathological responses. Excessive vascular smooth muscle cell (VSMC) proliferation is associated with the pathophysiology of atherosclerosis and restenosis post-angioplasty. Thus, drug targeting of pathological VSMC growth may be a suitable therapeutic intervention in vascular proliferative diseases. 2. In the present study, we investigated the mechanisms underlying VSMC growth arrest induced by the pharmacological agent PCA-4230. Addition of PCA-4230 to cultured VSMCs blocked the induction of cyclin D1 and cyclin A expression normally seen in serum-restimulated cells. Moreover, PCA-4230 inhibited cyclin-dependent kinase 2 (CDK2) activity and abrogated hyperphosphorylation of the retinoblastoma (Rb) gene product. Similarly, PCA-4230-dependent growth arrest of transformed cell lines correlated with reduced level of cyclin D1 protein and inhibition of CDK2 activity. Consistent with these findings, PCA-4230 repressed serum-inducible cyclin A promoter activity, and overexpression of either cyclin D1 or E2F1 efficiently circumvented this inhibitory effect. Importantly, adenovirus-mediated overexpression of E2F1 restored S-phase entry in PCA-4230-treated VSMCs, demonstrating that PCA-4230 represses cyclin A gene expression and VSMC growth via inhibition of the cyclin D1/E2F pathway. 3. Because of its ability to inhibit the growth of human VSMCs and transformed cell lines, future studies are warranted to assess whether PCA-4230 may be a suitable therapeutic intervention for the treatment of hyperproliferative disorders, including cardiovascular disease and cancer.

Role of Sp1 in the induction of p27 gene expression in vascular smooth muscle cells in vitro and after balloon angioplasty.

-The abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in atherosclerosis and restenosis. Although several studies have implicated the growth inhibitory protein p27(Kip1) (p27) in the control of myocyte growth and hypertrophy, little is known about the molecular mechanisms that regulate p27 expression in the cardiovascular system. In the present study, we demonstrate the interaction of the transcription factor Sp1 with 2 GC-rich sequences within the p27 promoter in cultured VSMCs. Importantly, point mutations that disrupted Sp1 binding markedly reduced p27 promoter activity, demonstrating that Sp1 is required for efficient p27 gene transcription in cultured VSMCs. Because p27 expression is upregulated after balloon angioplasty, we investigated Sp1 expression and activity in control and balloon-injured rat carotid arteries to assess the role of Sp1 as a physiological regulator of p27 expression. Although immunohistochemical analysis disclosed Sp1 protein expression in both control and balloon-injured arteries, a high level of Sp1 DNA-binding activity was found only in response to balloon angioplasty. Collectively, these results demonstrate that Sp1 is essential for maximum p27 promoter activity in VSMCs and suggest that posttranslational induction of Sp1 DNA-binding activity contributes to the induction of p27 expression and VSMC growth arrest at late time points after balloon angioplasty.

Mechanisms and implications of the age-associated decrease in DNA repair capacity.

Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6-4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6-4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.

Kinesin participates in melanosomal movement along melanocyte dendrites.

Movement of melanosomes along melanocyte dendrites is necessary for the transfer of melanin pigment from melanocytes to basal and suprabasal keratinocytes, an event critical to epidermal photoprotection and maintenance of normal skin color. Recent murine data suggest that in melanocyte dendrites the microtubule-associated melanosome movement is bidirectional and that actin-associated myosin V secures the peripheral melanosomes, preparing them to be transferred to surrounding keratinocytes. We now report that human melanocytes express high levels of kinesin, a molecule that participates in microtubule-associated transport of organelles in other cell types, and that ultrastructurally kinesin molecules are closely associated with melanosomes. To determine whether kinesin participates in melanosomal transport, cultured melanocytes were treated with sense or antisense oligonucleotides complementary to kinesin heavy chain sequences. Antisense oligonucleotides decreased kinesin protein levels and inhibited the bidirectional movement of the melanosomes, promoting their backward movement. Furthermore, guinea pigs were exposed to ultraviolet B irradiation, known to enhance transport of melanosomes from melanocytes to epidermal keratinocytes, and then were treated with kinesin sense or antisense oligonucleotides. The areas that were treated with kinesin antisense oligonucleotides showed significantly less pigmentation clinically and histologically than control (sense) oligonucleotide-treated areas. As observed ultrastructurally, in antisense-treated areas melanosomes remained in melanocyte dendrites but over several days were not transferred to the surrounding keratinocytes. Our study supports a major role for kinesin in microtubule-associated anterograde melanosomal transport in human melanocyte dendrites.

Thymidine dinucleotide mimics the effect of solar simulated irradiation on p53 and p53-regulated proteins.

The tumor suppressor protein p53 participates in DNA repair and cell cycle regulation in response to injuries like ultraviolet (UV) irradiation. We have previously reported that the thymidine dinucleotide (pTpT), a common target for DNA photoproduct formation by UV light, mimics many effects of UV irradiation in cultured skin-derived cells, at least in part through the activation of p53. In this report we compare the effects of solar-simulated irradiation and pTpT on p53 and p53-regulated proteins involved in cellular growth arrest and DNA repair in cultured human dermal fibroblasts. We find that, like UV irradiation, pTpT increases the levels of p53, p21, and proliferating-cell nuclear antigen. The magnitude and time course of the inductions are UV dose dependent and consistent with known regulatory interactions among these nuclear proteins. These data confirm and expand previous studies of UV effects on nuclear proteins involved in cell cycle regulation and DNA repair. Our observations suggest that such protective effects can also be induced by pTpT in the absence of initial DNA damage, rendering cells more capable of responding to subsequent DNA damage.