PAX3 in neuroblastoma: oncogenic potential, chemosensitivity and signalling pathways.

Transcription factor PAX3/Pax3 contributes to diverse cell lineages during embryonic development and is important in tumourigenesis. We found that PAX3 is re-expressed in neuroblastoma and malignant neuroblastic (N-type) neuroblastoma cells had significantly higher PAX3 protein expression than their benign substrate-adherent (S-type) counterparts. Knock-down of PAX3 expression by siRNA transfection resulted in persistent cell growth inhibition in both types of neuroblastoma cell, owing to G1 cell cycle arrest and progressive apoptosis. Inhibition of PAX3 expression significantly decreased the attachment of S-type SH-EP1 cells to extra-cellular matrix proteins, fibronectin, laminin and collagen IV. Migration and invasion of both neuroblastoma cell types were markedly reduced after PAX3 down-regulation. PAX3 knock-down significantly augmented the cytotoxic effect of chemotherapeutic agents, etoposide, vincristine and cisplatin, commonly used to treat neuroblastoma. Microarray analyses revealed that particularly signalling pathways involving cell cycle, apoptosis, cell adhesion, cytoskeletal remodelling and development were altered by PAX3 down-regulation. Changes in PAX3 downstream genes identified by microarray analyses were validated in 47 genes by quantitative PCR. These novel findings lead us to propose that PAX3 might contribute to oncogenic characteristics of neuroblastoma cells by regulating a variety of crucial signalling pathways.

The morphological postnatal development of the testis of the Nubian bucks.

This study was designed to monitor the morphological development of the reproductive tract of the Nubian bucks in relation to puberty. Thirty-two Nubain male kids were used in the study. The animals were slaughtered at intervals of 2 weeks starting from 1 day old up to 24 weeks of age. Tissue samples were obtained from the testes and processed for ultrastructural studies. The boundary tissue of the newly forming seminiferous tubule adhered closely to the basal lamina. It consisted of a single continuous layer of myoid cells, the outer surface of which was covered by scattered fibroblasts. The ultrastructural study of the boundary of the seminiferous tubule revealed that it consisted of three layers; inner fibrous, middle and outer cellular. The seminiferous tubules at week one were lined by two layers of epithelia; spermatogonia and Sertoli cells in the basal layer, and primary spermatocytes in the second layer. A gradual increase in the diameter of the tubules and epithelial height continued to increase with age. Furthermore, spermatocytes number showed an increase with age. In conclusion, based on the appearance of spermatozoa in the lumina of the seminiferous tubules, puberty age was achieved between weeks 18 and 20.

PAX3 and PAX3-FKHR promote rhabdomyosarcoma cell survival through downregulation of PTEN.

PAX3 or PAX3-FKHR expression is implicated in cell transformation and tumourigenesis. Here, C2C12 myoblasts were transfected with a sense Pax3 vector and a pTet-On system to induce Pax3 expression, whereas to downregulate PAX3-FKHR, Rh18 was transfected with an antisense Pax3 with a pTet-On system. The inhibition of PAX3-FKHR in Rh18 induced upregulation of PTEN. Decreased resistance to apoptosis and increased transformation ability were observed in the Rh18 cells with PAX3-FKHR downregulation. Conversely, Pax3 induction in C2C12 cells downregulated the expression of PTEN and p27(Kip1). These results indicate that the involvement of PAX3 and PAX3-FKHR in rhabdomyosarcoma tumourigenesis may be through downregulation of PTEN tumour suppressor gene, affecting the PTEN/AKT survival pathway.

Expression of signaling molecules associated with apoptosis in human ischemic stroke tissue.

There is growing evidence that, because of the highly significant differences in gene activation/protein expression between animal models of stroke and stroke patients, the current treatment strategies based on animal stroke models have been unsuccessful. Therefore, it is imperative that the pathobiology of human stroke be studied. As a first step here, Western blotting and immunohistochemistry were employed to examine expression and tissue localization of key apoptotic proteins in infarct and peri-infarcted (penumbra) from grey and white matter in human postmortem tissue of 18 patients who died between 2 and 37 d after stroke caused by large vessel disease. The contralateral hemisphere was used as a control. JNK1, JNK2, and p53 were upregulated in the majority of samples, whereas Bcl-2, caspase-3, active caspase-3, phosphorylated p53 (p-p53), phosphorylated JNK1 (p-JNK1), and phosphorylated JNK2 (p-JNK2) were upregulated in approximately half of the samples. JNK1 expression was positively correlated with JNK2 expression in grey and white matter infarct and penumbra, whereas active caspase-3 levels were positively correlated with p-JNK2 levels in grey and white matter infarct. Using indirect immunoperoxidase staining of paraffin-embedded sections, active caspase-3 was found in infarcted neurons that co-localized with TUNEL-positive cells. p-JNK localization in the nuclei of TUNELpositive cells with the morphological appearance of neurons from infarct and penumbra was also demonstrated. The use of Kaplan Meier survival data demonstrated that the presence of Bcl-2 in penumbra of grey matter correlated significantly with shorter survival (p = 0.006). In conclusion, the present study has identified significantly altered expression of apoptotic proteins in human stroke tissue and shown that the presence of Bcl-2 in penumbra of grey matter has prognostic value. It is tempting to suggest that further studies of apoptotic proteins in human stroke may lead to identification of novel targets for drug discovery.

Changes in hyaluronan production and metabolism following ischaemic stroke in man.

The extent of recovery from stroke is dependent on the survival of neurons, particularly in peri-infarcted regions. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion and better recovery. Hyaluronan (HA) is an important component of the brain extracellular matrix and a regulator of cellular differentiation, migration, proliferation and angiogenesis. We have found that the production of total HA and low molecular mass 3-10 disaccharides of HA (o-HA) was increased in post-mortem tissue and in the serum of patients 1, 3, 7 and 14 days (peaking at 7 days) after ischaemic stroke. Hyaluronidase activity was also increased in serum samples (peaking after 3 days), which might explain the subsequent increase in o-HA. Affinity-histochemical staining was performed using a HA-specific biotinylated binding protein, and it showed enhanced deposition of HA in blood vessels and intracellularly as well as in the nuclei of peri-infarcted neurons. Western blotting and immunohistochemistry demonstrated upregulation of HA synthases (HAS1 and 2) and hyaluronidases (HYAL1 and 2) in inflammatory cells from both stroke and peri-infarcted regions of the brain. HYAL1 was upregulated in microvesssels and intracellularly in neurons, whilst HAS2 became translocated into the nuclei of neurons in peri-infarcted areas. Receptor for HA-mediated motility was observed intracellularly and in the nuclei of neurons, in the tunica media of larger blood vessels and in the endothelial cells of microvessels in stroke-affected tissue, whilst expression of other receptors for HA, CD44 and tumour necrosis factor-stimulated gene 6 (TSG-6) were mainly increased in infiltrating mononuclear cells from inflammatory regions. The data presented here demonstrate that HA breakdown is a feature of the acute stage of stroke injury. Increased o-HA production soon after stroke may be detrimental through enhancement of the inflammatory response, whilst activation of HA and/or o-HA-induced cellular signalling pathways in neurons and microvessels may impact on the remodelling process by stimulating angiogenesis and revascularization, as well as the survival of susceptible neurons.

Perioperative morbidity of laparoscopic cryoablation of small renal masses with ultrathin probes: a European multicentre experience.

BACKGROUND: Low morbidity has been advocated for cryoablation of small renal masses. OBJECTIVES: To assess negative perioperative outcomes of laparoscopic renal cryoablation (LRC) with ultrathin cryoprobes and patient, tumour, and operative risk factors for their development. DESIGN, SETTING, AND PARTICIPANTS: Prospective collection of data on LRC in five centres. INTERVENTION: LRC. MEASUREMENTS: Preoperative morbidity was assessed clinically and the American Society of Anaesthesiologists (ASA) score was assigned prospectively. Charlson Comorbidity Index (CCI) and Charlson-Age Comorbidity Index (CACI) scores were retrospectively assigned. Negative outcomes were prospectively recorded and defined as any undesired event during the perioperative period, including complications, with the latter classed according to the Clavien system. Patient, tumour, and operative variables were tested in univariate analysis as risk factors for occurrence of negative outcomes. Significant variables (p<0.05) were entered in a step-forward multivariate logistic regression model to identify independent risk factors for one or more perioperative negative outcomes. The confidence interval was settled at 95%. RESULTS AND LIMITATIONS: There were 148 procedures in 144 patients. Median age and tumour size were 70.5 yr (range: 32-87) and 2.6 cm (range: 1.0-5.6), respectively. A laparoscopic approach was used in 145 cases (98%). Median ASA, CCI, and CACI scores were 2 (range: 1-3), 2 (range: 0-7), and 4 (range: 0-11), respectively. Comorbidities were present in 79% of patients. Thirty negative outcomes and 28 complications occurred in 25 (17%) and 23 (15.5%) cases, respectively. Only 20% of all complications were Clavien grade > or = 3. Multivariate analysis showed that tumour size in centimetres, the presence of cardiac conditions, and female gender were independent predictors of negative perioperative outcomes occurrence. Receiver operator characteristic curve confirmed the tumour size cut-off of 3.4 cm as an adequate predictor of negative outcomes. CONCLUSIONS: Perioperative negative outcomes and complications occur in 17% and 15.5%, respectively, of cases treated by LRC with multiple ultrathin needles. Most of the complications are Clavien grade 1 or 2. The presence of cardiac conditions, female gender, and tumour size are independent prognostic factors for the occurrence of a perioperative negative outcome.

Pax genes in embryogenesis and oncogenesis.

The paired box genes are a family of nine developmental control genes, which in human beings (PAX) and mice (Pax) encode nuclear transcription factors. The temporal and spatial expressions of these highly conserved genes are tightly regulated during foetal development including organogenesis. PAX/Pax genes are switched off during the terminal differentiation of most structures. Specific mutations within a number of PAX/Pax genes lead to developmental abnormalities in both human beings and mice. Mutation in PAX3 causes Waardenburg syndrome, and craniofacial-deafness-hand syndrome. The Splotch phenotype in mouse exhibits defects in neural crest derivatives such as, pigment cells, sympathetic ganglia and cardiac neural crest-derived structures. The PAX family also plays key roles in several human malignancies. In particular, PAX3 is involved in rhabdomyosarcoma and tumours of neural crest origin, including melanoma and neuroblastoma. This review critically evaluates the roles of PAX/Pax in oncogenesis. It especially highlights recent advances in knowledge of how their genetic alterations directly interfere in the transcriptional networks that regulate cell differentiation, proliferation, migration and survival and may contribute to oncogenesis.

Investigation of downstream target genes of PAX3c, PAX3e and PAX3g isoforms in melanocytes by microarray analysis.

PAX3 encodes a transcription factor, which with Zic1 is necessary for induction of the neural crest during early embryonic development. There are 7 human PAX3 isoforms (a-h). PAX3e is the full length isoform comprising 10 exons. PAX3c comprises 8 exons plus 5 codons of intron 8, while PAX3g has a truncated transactivation domain. Previous studies by us indicated that these isoforms have different activities in melanocytes in vitro. In this study, a mouse gene oligo array ( approximately 7.5 k oligos), from the Human Genome Mapping Project (HGMP) Resource Centre, was used to screen for alterations in downstream gene expression in PAX3c, PAX3e and PAX3g melanocyte transfectants, compared with empty vector controls. The data analyses identified 109 genes up or downregulated, at least 2-fold, and involved in cell differentiation, proliferation, migration, adhesion, apoptosis and angiogenesis. Semi-quantitative RT-PCR and Western blotting confirmed the changes identified by microarrays for several putative targets of PAX3, including Met, MyoD and Muc18, and previously undescribed targets, including Dhh, Fgf17, Kitl and Rac1. Thus, our data reveal that PAX3 isoforms regulate distinct but overlapping sets of genes in melanocytes in vitro.

Functional analysis of alternative isoforms of the transcription factor PAX3 in melanocytes in vitro.

Transcription factor PAX3 has seven isoforms of which PAX3c has been studied extensively whereas the functions of the other isoforms are less well known. Here, we found that PAX3 isoforms in a stable transfection system have different biological functions in mouse melanocytes in vitro. PAX3a and PAX3b had negative effects on melanocyte proliferation but had no discernable effect on melanocyte growth in soft agar. PAX3a did not affect cell migration and apoptosis but PAX3b reduced migration and accelerated apoptosis. PAX3c and PAX3d promoted cell proliferation, migration, transformation, and survival. PAX3e reduced melanocyte growth; transformation and migration were unchanged and apoptosis was increased in vitro. PAX3g did not influence cell proliferation or apoptosis. Cells expressing PAX3g were able to grow in soft agar but migration was reduced. PAX3h increased cell proliferation, migration, survival, and transformation. These functional studies have advanced our understanding of the effects of PAX3 isoforms in melanocytes and their potential contribution in tumorigenesis.

Expression of PAX 3 alternatively spliced transcripts and identification of two new isoforms in human tumors of neural crest origin.

The developmental gene PAX 3 is expressed in the early embryo in developing muscle and elements of the nervous system, including the brain. Since no one has investigated the expression of the isoforms of PAX 3 in the neuroectodermal tumors melanoma and small cell lung cancer (SCLC), we have carried out a comprehensive screening for the expression of the isoforms PAX 3a-e using RT-PCR in human melanoma cell lines, primary human ocular and secondary cutaneous melanomas. We have identified 2 new isoforms of PAX 3, g and h, which we have isolated, cloned and sequenced. Sets of primers for each isoform were designed and their specificity was confirmed by sequence analysis of the products. The isoforms PAX 3a-e were detected in all human cutaneous melanoma cell lines (8/8), but only PAX 3c (1/2) and PAX 3d (2/2) in ocular melanoma cell lines. The same PAX 3 isoforms were detected in more than 80% of human cutaneous melanomas: PAX 3a and b (15/17), PAX 3c (14/17), PAX 3d (16/17) and PAX 3e (15/17). In contrast the results for 7 SCLC cell lines were PAX 3a (0/7), PAX 3b (1/7), PAX 3c (3/7), PAX 3d (6/7), PAX 3e (2/7); 8/8 cutaneous melanoma cell lines and 8/8 ocular melanoma tissues, together with 14/17 cutaneous melanoma tissues screened, expressed the new isoform PAX 3g. All 8 cutaneous melanoma cell lines expressed PAX 3h, but it was not detectable in any of the tumor tissues (0/20). Neither of the 2 ocular melanoma cell lines expressed the 2 new isoforms. Comparison of the different amplicon staining intensities on a gel suggests that PAX 3c and PAX 3d are the predominant transcripts expressed, with relatively low expression of PAX 3e and PAX 3h. We propose that these and the 2 new isoforms we have discovered may be important in oncogenesis and differential diagnosis of melanomas or SCLC.