SATB2 enhances migration and invasion in osteosarcoma by regulating genes involved in cytoskeletal organization.

Osteosarcoma (OS) is the most common malignant bone tumor and the majority of recurrences are due to metastasis. However, the molecular mechanisms that regulate OS metastatic spread are largely unknown. In this study, we report that special AT-rich-binding protein 2 (SATB2) is highly expressed in OS cells and tumors. Short hairpin RNA-mediated knockdown of SATB2 (sh-SATB2) decreases migration and invasion of OS cells without affecting proliferation or viability. Microarray analysis identified genes that were differentially regulated by SATB2 including the actin-binding protein Epithelial Protein Lost In Neoplasm (EPLIN), which was upregulated in sh-SATB2 cells. Silencing EPLIN rescues the decreased invasion observed in sh-SATB2 cells. Pathway analyses of SATB2-regulated genes revealed enrichment of those involved in cytoskeleton dynamics, and increased stress fiber formation was detected in cells with SATB2 knockdown. Furthermore, sh-SATB2 cells exhibit increased RhoA, decreased Rac1 and increased phosphorylation of focal adhesion kinase (FAK) and paxillin. These findings identify SATB2 as a novel regulator of OS invasion, in part via effects on EPLIN and the cytoskeleton.

Regulatory feedback loop between TP73 and TRIM32.

The p73 transcription factor is one of the members of the p53 family of tumor suppressors with unique biological functions in processes like neurogenesis, embryonic development and differentiation. For this reason, p73 activity is tightly regulated by multiple mechanisms, including transcription and post-translational modifications. Here, we identified a novel regulatory loop between TAp73 and the E3 ubiquitin ligase tripartite motif protein 32 (TRIM32). TRIM32, a new direct p73 transcriptional target in the context of neural progenitor cells, is differentially regulated by p73. Although TAp73 binds to the TRIM32 promoter and activates its expression, TAp73-induced TRIM32 expression is efficiently repressed by DNp73. TRIM32 in turn physically interacts with TAp73 and promotes its ubiquitination and degradation, impairing p73-dependent transcriptional activity. This mutual regulation between p73 and TRIM32 constitutes a novel feedback loop, which might have important implications in central nervous system development as well as relevance in oncogenesis, and thus emerges as a possible therapeutic target.

Late mortality after hematopoietic SCT for a childhood malignancy.

Hematopoietic SCT (HSCT) has been used as a curative therapy for pediatric malignancies. Survivors of HSCT are at risk for disease recurrence, late morbidity and mortality. We assessed late mortality (≥2 years post-HSCT) in a population-based cohort of children who underwent HSCT for a malignancy. Mortality outcomes were determined by linking a clinical transplant database with the Canadian province of Ontario's pediatric cancer mortality files. Seven hundred and fifty-four children underwent HSCT (371 allogeneic, 383 autologous). Of the 479 (63.5%) who were alive ≥2 years post HSCT, 98 (20.5%) suffered a late death. Late mortality in the allogeneic HSCT group was 14.9% (median follow-up 10.0 years; range: 2.0-25.6 years), mainly due to relapse of the primary malignancy (64.7%). Chronic GVHD and second malignancies were not major causes of late mortality. A total of 25.5% suffered a late death following autologous HSCT (median follow-up 6.7 years; range: 2.0-22.2 years). Recurrence of the primary malignancy accounted for 87.5% of these deaths. Recurrence of the primary malignancy is the predominant cause of late mortality after HSCT. In contrast to studies of adult patients, non-relapse mortality is less common in children, and death due to chronic GVHD and secondary malignancies is uncommon.

Targeting the p53-family in cancer and chemosensitivity: triple threat.

The p53-family of transcription factors consists of three genes - p53, p63, and p73 - that share significant structural and functional similarities. Although these genes encode multiple variants that have opposing functions in cancer biology, the full-length, transactivating (TA) p53-family members are potent inducers of apoptosis and tumor suppression. Many anti-cancer agents, from traditional chemo- and radiation therapies to more recently developed small molecules, exert their effects by enhancing the anti-proliferative effects of p53 and TAp63/p73. In this review, we provide an overview of the regulatory pathways controlling the p53-family proteins as a framework for understanding p53-family targeted drug mechanisms. We will also summarize recent work on promising attempts to re-activate p53 in tumors. In addition, we will discuss how p63 and p73 - the two more recently discovered p53-family members - have affected drug discovery and how these two genes may also hold promise as drug targets for recent and future novel therapies. This review will emphasize how targeting multiple members of the family of p53 proteins is likely to provide an increased threat to the growth of cancer cells.

Resource implications of bilateral autologous breast reconstruction--a single centre's seven year experience.

INTRODUCTION AND AIMS: Since the recent introduction of ''Payment by Results'' as part of NHS financial reforms, it has been noted that there is an imbalance between allocated Healthcare Resource Group tariffs and actual resource use for certain procedures. This study was undertaken to assess the impression that bilateral breast reconstruction using autologous flaps is under-funded. MATERIAL AND METHODS: Patients who underwent bilateral flap breast reconstruction following mastectomy between 2000 and 2006 at Addenbrooke's University Hospital were identified. Resource cost analysis for each patient was based on the following parameters: number of operating consultants, theatre running costs, and length of hospital stay. The estimated hospital costs were then compared to the national tariff for the Healthcare Resource Group ''Complex Breast Reconstruction using Flaps''. KEY RESULTS: Over the 7-year period 24 patients underwent bilateral flap breast reconstruction (7 paired latissimus dorsi and 17 paired abdominal flaps). The mean operative time was 9.4h (£4.5/min), the mean hospital stay was 10 days (£150/day) and ten patients required 2 consultants (£34/h) operating. The average total cost equated to £5 492. CONCLUSION: The allocated tariff of £4 053 is insufficient, even before the inclusion of hidden costs. Bilateral free flap breast reconstructions are grossly under-funded at present. With increasing financial pressures on NHS Trusts there may be a drive towards simpler operations, which receive proportionally greater remuneration.

Chemotherapy induces NEDP1-mediated destabilization of MDM2.

MDM2 is an E3 ligase that promotes ubiquitin-mediated destruction of p53. Cellular stresses such as DNA damage can lead to p53 activation due in part to MDM2 destabilization. Here, we show that the stability of MDM2 is regulated by an ubiquitin-like NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization concomitant with p53 activation. Concordantly, RNAi-mediated knockdown of endogenous NEDP1 blocked diminution of MDM2 levels and increased chemoresistance of tumor cells. These findings unveil the regulation of MDM2 stability through NEDP1 as a common molecular determinant governing chemotherapy-induced p53-dependent cell death.

Cyclooxygenase inhibitors differentially modulate p73 isoforms in neuroblastoma.

p73 encodes multiple functionally distinct isoforms. Proapoptotic TAp73 isoforms contain a transactivation (TA) domain, and like p53, have tumor suppressor properties and are activated by chemotherapies to induce cell death. In contrast, antiapoptotic DeltaNp73 isoforms lack the TA domain and are dominant-negative inhibitors of p53 and TAp73. DeltaNp73 proteins are overexpressed in a variety of tumors including neuroblastoma. Thus, identification of drugs that upregulate TAp73 and/or downregulate DeltaNp73 represents a potential therapeutic strategy. Here, we report that cyclooxygenase (COX) inhibitors induce apoptosis independent of p53, and differentially modulate endogenous p73 isoforms in neuroblastoma and other tumors. COX inhibitor-mediated apoptosis is associated with the induction of TAp73beta and its target genes. COX inhibitors also downregulate the alternative-spliced DeltaNp73(AS) isoforms, Deltaexon2 and Deltaexon2/3. Furthermore, forced expression of DeltaNp73(AS) results in diminished apoptosis in response to the selective COX-2 inhibitor celecoxib. Celecoxib-mediated downregulation of DeltaNp73(AS) is associated with decreased E2F1 levels and diminished E2F1 activation of the p73 promoter. These results provide the first evidence that COX inhibitors differentially modulate p73 isoforms leading to enhanced apoptosis, and support the potential use of COX inhibitors as novel regulators of p73 to enhance chemosensitivity in tumors with deregulated E2F1 and in those with wild-type (wt) or mutant p53.

Abdominoplasty as an adjunct to the management of peri-Caesarian section necrotising fasciitis.

Necrotising fasciitis is a rare but potentially lethal condition, often requiring extensive soft tissue debridement and complex reconstructive surgery. The disease has been noted to complicate Caesarian section wounds, and our department has recently managed three such patients. They all required extensive abdominal wall debridements which would traditionally be closed initially by split skin grafting. We report on the clinical course of three patients, two of whom had their defects closed successfully by abdominoplasty without recourse to initial skin grafting.

HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73 function.

Nutlin-3, a small molecule inhibitor, activates p53 by disrupting p53-HDM2 association. In this study, we found that Nutlin-3 suppressed cell growth and induced apoptosis in the absence of wild-type p53, suggesting a p53-independent mechanism for Nutlin-3-induced cell death. Like p53, its homolog p73 transactivates proapoptotic genes and induces cell death. Since HDM2, a key negative regulator of p53, also binds to and inhibits p73, we asked whether p73 could mediate Nutlin-3-induced apoptosis. We demonstrate that Nutlin-3 inhibits endogenous binding between the proapoptotic p73 isoform TAp73alpha and HDM2 in p53-null cells. Dissociation of p73 and HDM2 leads to increased p73 transcriptional activity with upregulation of p73 target genes noxa, puma and p21, as well as enhanced apoptosis. p73 knockdown by siRNA results in rescue of Nutlin-3-treated cells, indicating that Nutlin-3-induced apoptosis is, at least in part, p73 dependent. In addition, Nutlin-3 treatment increases TAp73alpha protein levels with prolongation of p73 half-life. These results provide the first evidence that Nutlin-3 disrupts endogenous p73-HDM2 interaction and enhances the stability and proapoptotic activities of p73 and thus, provides a rationale for the use of Nutlin-3 in the large number of human tumors in which p53 is inactivated.

Cyclooxygenase inhibitors modulate the p53/HDM2 pathway and enhance chemotherapy-induced apoptosis in neuroblastoma.

Cyclooxygenase-2 (COX-2) is upregulated in many tumors including neuroblastoma, and its overexpression has been implicated in resistance to p53-dependent apoptosis. Although p53 is rarely mutated in neuroblastoma, the p53 protein is rendered inactive via several mechanisms including sequestration in the cytoplasm. Here, we show that COX inhibitors inhibit the growth of neuroblastoma and when combined with low doses of chemotherapy, exert synergistic effects on neuroblastoma cells. Following COX inhibitor treatment, HDM2, which targets p53 for ubiquitin-mediated degradation, is downregulated, resulting in an attenuation of p53 ubiquitination and an increase in p53 half-life. The level of HDM2 phosphorylation at ser166, which influences both HDM2 and p53 subcellular distribution, is markedly diminished in response to COX inhibitors and is associated with increased p53 nuclear localization. Combining COX inhibitors with low-dose chemotherapy potentiates apoptosis and p53 stability, nuclear localization, and activity. p53 knockdown by siRNA resulted in the rescue of COX-inhibitor-treated cells, indicating that COX inhibitor-induced apoptosis is, at least in part, p53-dependent. Taken together, these results provide the first evidence that COX inhibitors enhance chemosensitivity in neuroblastoma via downregulating HDM2 and augmenting p53 stability and nuclear accumulation.

Role of the newer p53 family proteins in malignancy.

The most recently identified members of the p53 family, p63 and p73, share certain structural and functional similarities with p53. Both p63 and p73 can bind to canonical p53-DNA-binding sites, transactivate the promoters of known p53 target genes and induce apoptosis. Despite these similarities there are many important differences. In contrast to p53, p63 and p73 give rise to multiple distinct protein isoforms that have different functional properties. Upstream signaling pathways involved in the activation of p63 and p73 differ from those involved in p53 activation. Only a subset of the DNA damaging agents that induce p53 can induce p73. Cellular and viral oncoproteins can discriminate between p53 and the newer family members. In addition, the levels of p63 and p73 are affected by certain states of cellular differentiation. Finally, it is becoming clear that the newest members of the p53 family are not classical tumor suppressor genes. In contrast to the high prevalence of p53 mutations in human cancers, p63 and p73 mutations are rare. Indeed, levels of p73 increase during malignant progression. In addition, unlike p53-/- mice, mice lacking p63 and p73 do not develop tumors, but instead have significant developmental abnormalities. Mutations in p63 have also been detected in humans with the ectodermal dysplastic syndrome EEC. Further studies are required to determine whether qualitative or quantitative differences in the expression of p63 and p73 isoforms are important in the development of human cancers.

A common polymorphism acts as an intragenic modifier of mutant p53 behaviour.

The p73 protein, a homologue of the tumour-suppressor protein p53, can activate p53-responsive promoters and induce apoptosis in p53-deficient cells. Here we report that some tumour-derived p53 mutants can bind to and inactivate p73. The binding of such mutants is influenced by whether TP53 (encoding p53) codon 72, by virtue of a common polymorphism in the human population, encodes Arg or Pro. The ability of mutant p53 to bind p73, neutralize p73-induced apoptosis and transform cells in cooperation with EJ-Ras was enhanced when codon 72 encoded Arg. We found that the Arg-containing allele was preferentially mutated and retained in squamous cell tumours arising in Arg/Pro germline heterozygotes. Thus, inactivation of p53 family members may contribute to the biological properties of a subset of p53 mutants, and a polymorphic residue within p53 affects mutant behaviour.

Viral oncoproteins discriminate between p53 and the p53 homolog p73.

p73 is a recently identified member of the p53 family. Previously it was shown that p73 can, when overproduced in p53-defective tumor cells, activate p53-responsive promoters and induce apoptosis. In this report we describe the generation of anti-p73 monoclonal antibodies and confirm that two previously described p73 isoforms are produced in mammalian cells. Furthermore, we show that these two isoforms can bind to canonical p53 DNA-binding sites in electrophoretic mobility shift assays. Despite the high degree of similarity between p53 and p73, we found that adenovirus E1B 55K, simian virus 40 T, and human papillomavirus E6 do not physically interact with p73. The observation that viral oncoproteins discriminate between p53 and p73 suggests that the functions of these two proteins may differ under physiological conditions. Furthermore, they suggest that inactivation of p73 may not be required for transformation.

Neuropathy in non-freezing cold injury (trench foot).

Non-freezing cold injury (trench foot) is characterized, in severe cases, by peripheral nerve damage and tissue necrosis. Controversy exists regarding the susceptibility of nerve fibre populations to injury as well as the mechanism of injury. Clinical and histological studies (n = 2) were conducted in a 40-year-old man with severe non-freezing cold injury in both feet. Clinical sensory tests, including two-point discrimination and pressure, vibration and thermal thresholds, indicated damage to large and small diameter nerves. On immunohistochemical assessment, terminal cutaneous nerve fibres within the plantar skin stained much less than in a normal control whereas staining to von Willebrand factor pointed to increased vascularity in all areas. The results indicate that all nerve populations (myelinated and unmyelinated) were damaged, possibly in a cycle of ischaemia and reperfusion.

Cold intolerance following peripheral nerve injury. Natural history and factors predicting severity of symptoms.

Cold intolerance can be severe and debilitating following injury to the hand. Little is known about its natural history and factors predicting symptom severity. We looked retrospectively at upper limb peripheral nerve injuries over a 12-year period. Information was obtained using a patient questionnaire and patient records. The incidence of cold intolerance was 83%. In 48% the onset of symptoms was within 1 month of the initial injury. At a mean follow-up of 51 months improved symptoms were reported by 21%, while 18% deteriorated. Patients were more likely to develop cold intolerance if they smoked and less likely if they suffered a sharp injury. A score defining the severity of cold induced symptoms, based on the information collected, was calculated for each patient. Significantly increased severity was associated with complete nerve division, median and ulnar nerve division and an associated vessel injury. Symptom improvement was significantly more likely in non-smokers and a deterioration most likely with a high severity score.

Nature and mechanism of peripheral nerve damage in an experimental model of non-freezing cold injury.

Non-freezing cold injury (NFCI), so called trench foot, is a condition characterised by a peripheral neuropathy, developing when the extremities are exposed for prolonged periods to wet conditions at temperatures just above freezing. Classically, military personnel are affected, with 14% of casualties in the Falklands conflict afflicted. Clinically, NFCI is characterised by a well-defined acute clinical picture and chronic sequelae. Little is known regarding the pathophysiology and treatment of this condition. Opinions vary as to the type of nerve fibres most susceptible to damage and proposed mechanisms of injury include direct axonal damage, ischaemia and ischaemia/reperfusion. A series of investigations has been performed to clarify which populations of nerve fibres are more susceptible to damage, and to elucidate the exact mechanism of nerve injury. An in vivo rabbit hind limb model, subjected to 16 h of cold immersion (1-2 degrees C), provided the basis of this study. Nerve specimens were examined by semi-thin sectioning for myelin fibre counts, by electron microscopy to assess the unmyelinated fibre population, and fine nerve terminals in plantar skin were assessed immunohistochemically. The results showed that large myelinated fibres were preferentially damaged, while small myelinated and unmyelinated fibres were relatively spared. Nerve damage was found to start proximally and extend distally with time. Serial temperature measurements identified a warm-cold interface in the upper tibial region of immersed limbs. As this was the initial site of injury, this suggested that a dynamic balance exists in the cold immersed limb between the protective effects of cooling and the damaging effects of ischaemia. The non-invasive technique of near infrared spectroscopy was used to measure changes in tissue oxygen supply and utilisation and blood volume. The findings supported the hypothesis that an interface is created at the site of initial nerve damage in the upper tibia, where cyclical ischaemia-reperfusion injury occurs.

Near infra-red spectroscopy: a non-invasive monitor of perfusion and oxygenation within the microcirculation of limbs and flaps.

Reliable early detection of adverse circulatory changes within a flap following free tissue transfer and early re-exploration are vital to minimise flap failure. Most surgeons rely on clinical assessment to monitor these changes but techniques such as plethysmography and laser Doppler have their advocates. These methods are limited however to measuring changes close to the surface. Near infra-red spectroscopy (NIRS) is a relatively new, non-invasive technique which allows continuous monitoring of concentration changes in oxy-, deoxy- and total haemoglobin (HbO2, Hb and HbT), as well as oxidised cytochrome aa3, through tissue up to 10 cm in depth. Information is provided on tissue oxygen supply, cellular oxygen utilisation, blood volume and perfusion status. A study has been performed in 10 rabbit hind limbs to assess the ability of NIRS to detect and distinguish between venous, arterial and total vascular occlusion. Clear patterns of change have been identified which allow rapid detection of vascular occlusion with accurate prediction of site. Arterial occlusions were characterised by an increase in Hb with a corresponding decrease in HbO2 and HbT. Venous occlusions resulted in an increase in HbT with relatively minor fluctuations in Hb and HbO2. Simultaneous occlusion of both artery and vein produced similar changes to those of arterial occlusion except that HbT decreased only minimally. These findings suggest that NIRS has a potentially useful role in the monitoring of free flaps, with the great advantage that perfusion can be measured to a considerable depth and information provided on the oxygenation profiles both accurately and non-invasively.