The expression of fibroblast growth factors and their receptors in the embryonic and neonatal mouse inner ear

Four different fibroblast growth factor receptors (FGFR) are known, three of which have splice variants (known as the b and c variants) in the FGF-binding domain, to give different patterns of sensitivity to the different FGFs. The expression of the b and c variants of the FGF receptors. together with the expression of the ligands FGF1. FGF2, FGF3, FGF7, FGF8b and FGF8c, was determined by quantitative reverse transcription-polymerase chain reaction in developing whole mouse inner ears, and in dissected components of the postnatal mouse inner ear. At embryonic age (E)10.5 days, when the otocyst is a simple closed sac, the receptor most heavily expressed was FGFR2b, relative to the postnatal day 0 level. Over the period E10.5-E12.5. during which the structures of the inner ear start to form, the expression of the different FGF receptors increased 10(2)-10(4) fold per unit of tissue, and there was a gradual switch towards expression of the 'c' splice variants of FGFR2 and FGFR3 rather than the 'b' variants. At E10.5, the ligands most heavily expressed, relative to the postnatal day 0 level, were FGF3, FGF8b and FGF8c. In the postnatal inner eat. the patterns of expression of receptors and ligands tended to be correlated, such that receptor variants were expressed in the same regions as the ligands that are known to activate them effectively. The neural/sensory region expressed high levels of FGFR3c, and high levels of the ligand FGF8b. The same area also expressed high levels of FGFR1b and FGFR2b, and high levels of FGF3. The lateral wall of the cochlea (including the stria vascularis and the spiral ligament) expressed high levels of FGFR1c and FGF1. 11 is suggested that the different FGF receptors and ligands are expressed in a spatially coordinated pattern to selectively program cochlear development. (C) 2001 Elsevier Science B.V. All rights reserved.

Transactivation-deficient p73 alpha (p73 Delta exon2) inhibits apoptosis and competes with p53

p73 has recently been identified as a structural and functional homolog of the tumor suppressor protein p53. Overexpression of p53 activates transcription of p53 effector genes, causes growth inhibition and induced apoptosis. We describe here the effects of a tumor-derived truncated transcript of p73 alpha (p73 Delta exon2) on p53 function and on cell death. This transcript, which lacks the acidic N-terminus corresponding to the transactivation domain of p53, was initially detected in a neuroblastoma cell line. Overexpression of p73 Delta exon2 partially protects lymphoblastoid cells against apoptosis induced by anti-Fas antibody or cisplatin. By cotransfecting p73 Delta exon2 with wild-type p53 in the p53 null line Saos 2, we found that this truncated transcript reduces the ability of wild-type p53 to promote apoptosis. This anti-apoptotic effect was also observed when p73 Delta exon2 was co-transfected with full-length p73 (p73 alpha). This was further substantiated by suppression of p53 transactivation of the effector gene p21-Waf1 in p73 Delta exon2 transfected cells and by inhibition of expression of a reporter gene under the control of the p53 promoter. Thus, this truncated form of p73 can act as a dominant-negative agent towards transactivation by p53 and p73 alpha, highlighting the potential implications of these findings for p53 signaling pathway. Furthermore, we demonstrate the existence of a p73 Delta exon2 transcript in a very significant proportion (46%) of breast cancer cell lines. However, a large spectrum of normal and malignant tissues need to be surveyed to determine whether this transdominant p73 variant occurs in a tumor-specific manner.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1·Mre11·Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(o)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(o)/G(1), blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(o)/G(1), and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEG-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta -dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(o)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms. (C) 2001 Elsevier Science Inc. All rights reserved.

A for stage III and IV squamous carcinoma of the head and neck: a Trans-Tasman Radiation Oncology Group Study

Purpose: The aims of this randomized controlled trial were to determine whether there were differences in the disease-free survival (DFS) and toxicity between conventional radiotherapy (CRT) and a continuous 3 week accelerated radiotherapy regimen (ART) in stage III and IV squamous cell carcinoma of the oral cavity, oropharynx, larynx and hypopharynx. Patients and methods: Patients from 14 centres throughout Australia and New Zealand were randomly assigned to either CRT, using a single 2 Gy/day to a dose of 70 Gy in 35 fractions in 49 days or to ART, using 1.8 Gy twice a day to a dose of 59.4 Gy in 33 fractions in 24 days. Treatment allocation was stratified for site and stage. The accrual began in 1991 and the trial was closed in 1998 when the target of 350 patients was reached. Results: The median potential follow-up time was 53 months (range, 14-101). The DFS at 5 years was 41% (95% CI, 33-50%) for ART and 35% (95% CI, 27-43%) for CRT (P = 0.323) and the hazard ratio was 0.87 in favour of ART (95% CI, 0.66-1.15). The 5-year disease-specific survival rates were 40% for CRT and 46% for ART (P = 0.398) and the loco-regional control was 47% for CRT vs. 52% for ART (P = 0.300). The respective hazard ratios were 0.88 (95% CI, 0.65-1.2) and 0.85 (0.62-1.16), favouring the accelerated arm. In the ART arm, confluent mucositis was more severe (94 vs. 71%; P < 0.001) and peaked about 3 weeks earlier than in the CRT arm, but healing appeared complete in all cases. There were statistically significant reductions in the probability of grade 2 or greater late soft tissue effects over time in the ART arm (P < 0.05), except for the mucous membrane where late effects were similar in both arms. Conclusions: Differences in DFS, disease-specific survival and loco-regional control have not been demonstrated. ART resulted in more acute mucosal toxicity, but this did not result in greater prolongation of the treatment time compared with the CRT arm. There were less late effects in the ART arm, with the exception of late mucosal effects. This trial has confirmed that tumour cell repopulation occurs during conventionally fractionated radiotherapy for head and neck cancer. However, it has also provided additional evidence that overall improvements in the therapeutic ratio using accelerated fractionation strategies are seriously constrained by the need to limit total doses to levels that do not exceed acute mucosal tolerance. The accelerated schedule tested has been shown in this trial to be an acceptable alternative to conventionally fractionated irradiation to 70 Gy. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.