Bioengineered lungs consisting of a decellularized lung scaffold that is repopulated with a patient’s own cells could provide desperately needed donor organs in the future. This approach has been tested in rats, and has been partially explored in porcine and human lungs. However, existing bioengineered lungs are fragile, in part because of their immature vascular structure. Herein, we report the application of adipose-derived stem/stromal cells (ASCs) for engineering the pulmonary vasculature in a decellularized rat lung scaffold. We found that pre-seeded ASCs differentiated into pericytes and stabilized the endothelial cell (EC) monolayer in nascent pulmonary vessels, thereby contributing to EC survival in the regenerated lungs. The ASC-mediated stabilization of the ECs clearly reduced vascular permeability and suppressed alveolar hemorrhage in an orthotopic transplant model for up to 3 h after extubation. Fibroblast growth factor 9, a mesenchyme-targeting growth factor, enhanced ASC differentiation into pericytes but overstimulated their proliferation, causing a partial obstruction of the vasculature in the regenerated lung. ASCs may therefore provide a promising cell source for vascular regeneration in bioengineered lungs, though additional work is needed to optimize the growth factor or hormone milieu for organ culture.

Defective DNA damage response (DDR) can result in genomic instability (GIN) and lead to the transformation into cancer. P53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DNA double strand breaks (DSBs) and rapidly form nuclear foci, the presence of 53BP1 foci can be considered as a cytologic marker for endogenous DSBs reflecting GIN. Although it has been proposed that GIN has a crucial role in the progression of thyroid neoplasms, the significance of GIN during thyroid tumorigenesis remains unclear, particularly in patients. We analyzed, therefore, the level of GIN, as detected with immunofluorescence of 53BP1, in 40 cases of resected thyroid tissues. This study demonstrated a number of nuclear 53BP1 foci in thyroid cancers, suggesting a constitutive activation of DDR in thyroid cancer cells. Because follicular adenoma also showed a few 53BP1 nuclear foci, GIN might be induced at a precancerous stage of thyroid tumorigenesis. Furthermore, high-grade thyroid cancers prominently exhibited an intense and heterogeneous nuclear staining of 53BP1 immunoreactivity, which was also observed in radiation-associated cancers and in mouse colonic crypts as a delayed response to a high dose ionizing radiation, suggesting increased GIN with progression of cancer. Thus, the present study demonstrated a difference in the staining pattern of 53BP1 during thyroid carcinogenesis. We propose that immunofluorescence analysis of 53BP1 expression can be a useful tool to estimate the level of GIN and, simultaneously, the malignant potency of human thyroid tumors. ' 2007 Wiley-Liss, Inc.Key words: thyroid cancer; genomic instability; 53BP1; ATM; immunofluorescence DNA damage response (DDR) genes, such as p53, are frequently mutated in human cancer. Defective DDR responses can thus result in genomic instability (GIN) and lead to the transformation of normal cells into cancer cells. Thyroid cancer has been shown to display aneuploidy, one form of GIN. 1 It has been proposed that GIN has a crucial role in the progression of thyroid neoplasms. 2 Thus, transfection of mutant HRAS V12 or mutant BRAF V600E induced GIN in a rat thyroid cell line, manifesting as loss of chromosomal material, mitotic bridge formation and misaligned chromosomes. 3-5 Recently, we found RET oncogene amplification in human thyroid cancers. 6 Oncogene amplification is common in solid tumors and correlates with a poor prognosis for patients with ovarian cancer (HER-2/neu), breast cancer (C-MYC, HER-2/neu), neuroblastoma (N-MYC), or small cell lung cancer (C-MYC). [7][8][9][10] In thyroid cancers, RET amplification correlated with radiation-induced and high-grade malignancy, providing further evidence for the involvement of GIN in tumor progression. 6 P53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins with C-terminal BRCT domains. 11,12 The ataxia-teleangiactasis mutated (ATM) DDR kinase is well known as a sensor molecule for DNA damage. Both...

Epidermal cells are the first cells to be exposed to environmental genotoxic agents such as ultraviolet and ionizing radiations, which induce DNA double strand breaks (DSB) and activate DNA damage response (DDR) to maintain genomic integrity. Defective DDR can result in genomic instability (GIN) which is considered to be a central aspect of any carcinogenic process. P53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DSB and rapidly form nuclear foci, the presence of 53BP1 nuclear foci can be considered as a cytological marker for endogenous DSB reflecting GIN. The levels of GIN were analyzed by immunofluorescence studies of 53BP1 in 56 skin tumors that included 20 seborrheic keratosis, eight actinic keratosis, nine Bowen's disease, nine squamous cell carcinoma, and 10 basal cell carcinoma. This study demonstrated a number of nuclear 53BP1 foci in human skin tumorigenesis, suggesting a constitutive activation of DDR in skin cancer cells. Because actinic keratosis showed a high DDR type of 53BP1 immunoreactivity, GIN seems to be induced at the precancerous stage. Furthermore, invasive cancers exhibited a high level of intense, abnormal 53BP1 nuclear staining with nuclear accumulation of p53, suggesting a disruption of DDR leading to a high level of GIN in cancer cells. The results of this study suggest that GIN has a crucial role in the progression of skin carcinogenesis. The detection of 53BP1 expression by immunofluorescence can be a useful histological marker to estimate the malignant potential of human skin tumors. (Cancer Sci 2008; 99: 946-951) T he skin is the primary barrier for humans against the external environment. Therefore, epidermal cells are the first cells to be exposed to physical and chemical genotoxic agents such as UV and IR. IR effectively induces DSB in normal cells and activates DDR pathways to maintain genomic integrity. DDR genes, such as p35, are frequently mutated in human cancer. Thus, defective DDR can result in GIN which is generally considered to be a central aspect of any carcinogenic process. (1,2) It has been shown that gamma irradiation induced skin tumors in mice. Most of the tumor-bearing mice showed a loss of the wild-type p53 allele. Since no skin tumor was found in wild-type p53 mice, this suggested a requirement of p53 loss in irradiation-induced skin carcinogenesis. (3) The incidence of skin cancer was reported to be elevated in atomic bomb survivors, which also suggested a radiation etiology in human skin carcinogenesis. (4) 53BP1 belongs to a family of evolutionarily conserved DDR proteins with C-terminal BRCT (BRCA1 C-terminus) domains. (5,6) The 53BP1 is a nuclear protein that rapidly localizes at the sites of DSB, and cooperatively activates p53 with other kinases. (7-12) Subsequently, activated p53 plays a critical role in cellular responses to genomic injury, such as cell cycle arrest, DNA repair, and apoptosis. (13,14) It has been well documented in vitro with immunofluorescen...

The effect of basic fibroblast growth factor (bFGF) was studied in radiation-induced apoptosis in rat jejunal crypt cells. Six-week-old male Wistar rats were administered 4 mg/kg bFGF intraperitoneally 25 h before receiving 8 Gy whole-body X rays. The jejunum was removed for analysis from time 0 to 120 h after irradiation. Villus length in control rats declined steadily until 72 h, while in bFGF-treated rats the villi were longer than in the controls until 48 h. Crypt lengths were similar to villi. bFGF treatment increased Ki-67-positive cells in the jejunal crypt at 0, 24 and 48 h. The treatment with bFGF reduced the number of apoptotic cells per jejunal crypt to 23% and 10% of the control values at 3 and 6 h, respectively, and increased numbers of mitotic cells significantly at 48 and 72 h. bFGF decreased the levels of TP53, CDKN1A, Puma and Cleaved caspase 3 at 3 h as detected by Western blot analyses. Our results suggest that bFGF protected against acute radiation-induced injury by suppressing the crypt apoptotic cells including the stem cells and promoted crypt cell proliferation. The inhibition of apoptosis thus might be related to suppression of the TP53 pathway.

Radiotherapy for malignant pelvic disease is often followed by acute radiation colitis (ARC). It has been reported that sucralfate treatment has a protective effect against ARC, though the mechanisms of action are unknown. The effects of sucralfate on X-ray radiation-induced apoptosis was studied at 4 Gy in the colonic crypt cells of rats. Sucralfate enemas given prior to radiation resulted in the following: (1) reduction in number of apoptotic colonic crypt cells; (2) reduction in number of caspase-3 positive cells; (3) decreases in p53 accumulation and p21 expression; (4) decreases of Bax/Bcl-2 ratio. The protective effects of sucralfate against ARC may be partially due to the suppression of radiation-induced apoptosis by way of p53 in the colon and the protection of the colonic epithelial stem cell region.