1: Mol Cancer Ther. 2005 Jun;4(6):996-1003. Transfection of melanoma cells with antisense PAX3 oligonucleotides additively complements cisplatin-induced cytotoxicity. He SJ, Stevens G, Braithwaite AW, Eccles MR. Department of Pathology, Dunedin School of Medicine, New Zealand. Advanced melanoma is difficult to treat, in part because of greater resistance to therapy compared with other cancer types. The mechanisms underlying this resistance are not well-understood. One factor that is reported to be involved in melanoma cell survival is PAX3, a transcription factor normally expressed during embryonic development, and which is critically required for development of neural crest-derivatives, including skin melanocytes. PAX3 expression is deregulated in primary melanomas and most melanoma cell lines. Here we have investigated whether targeting PAX3 expression in melanoma cell lines together with chemotherapeutic treatment increases susceptibility to therapeutic cell death. Using PAX3-specific antisense oligodeoxynucleotides (PAX3-AS) to treat melanoma cell lines in vitro, we showed dose-dependent reduction of proliferation of melanoma cells, and induction of apoptosis compared with control treatments. Induction of apoptosis was accompanied by the induction of active caspase-3 in UACC62 and M14 cells, and p53 protein in UACC62 cells. Treatment of melanoma cells with cisplatin induces DNA damage and cytotoxicity, which is thought to be via p53-dependent and -independent mechanisms. Treatment of either p53 mutant (M14) or wild-type (UACC62) melanoma cells with cisplatin, and varying doses of PAX3-AS, resulted in percentages of cells undergoing apoptosis equivalent to the sum of the individual treatments, irrespective of mutation status [e.g., UACC62, 43.8% (1 micromol/L PAX3-AS), 30.1% (20 micromol/L cisplatin), 69.6% (PAX3-AS + cisplatin); M14, 12.6% (1 micromol/L PAX3-AS), 41.5% (40 micromol/L cisplatin), 50.2% (PAX3-AS + cisplatin)]. These data suggest that treatment of melanoma cells with PAX3-AS complements cytotoxicity induced by cisplatin. PMID: 15956257 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Genes Dev. 2004 Nov 1;18(21):2614-26. Epub 2004 Oct 15. Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function. Keller C, Arenkiel BR, Coffin CM, El-Bardeesy N, DePinho RA, Capecchi MR. Division of Pediatric Hematology-Oncology and Department of Pediatrics, University of Utah, Salt Lake City, Utah 84112, USA. Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer for which outcomes are poor when the disease is advanced. Although well-developed mouse models exist for embryonal and pleomorphic rhabdomyosarcomas, neither a spontaneous nor a transgenic mouse model of alveolar rhabdomyosarcoma has yet been reported. We report the first mouse model of alveolar rhabdomyosarcoma using a conditional Pax3:Fkhr knock-in allele whose activation in late embryogenesis and postnatally is targeted to terminally differentiating Myf6-expressing skeletal muscle. In these mice, alveolar rhabdomyosarcomas occur but at low frequency, and Fkhr haploinsufficiency does not appear to accelerate tumorigenesis. However, Pax3:Fkhr homozygosity with accompanying Ink4a/ARF or Trp53 pathway disruption, by means of conditional Trp53 or Ink4a/ARF loss of function, substantially increases the frequencies of tumor formation. These results of successful tumor generation postnatally from a target pool of differentiating myofibers are in sharp contrast to the birth defects and lack of tumors for mice with prenatal and postnatal satellite cell triggering of Pax3:Fkhr. Furthermore, these murine alveolar rhabdomyosarcomas have an immunohistochemical profile similar to human alveolar rhabdomyosarcoma, suggesting that this conditional mouse model will be relevant to study of the disease and will be useful for preclinical therapeutic testing. PMID: 15489287 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Oncogene. 2003 Sep 11;22(39):7989-97. Paired-Box genes are frequently expressed in cancer and often required for cancer cell survival. Muratovska A, Zhou C, He S, Goodyer P, Eccles MR. Department of Pathology, Dunedin School of Medicine, University of Otago, PO Box 913, Dunedin, New Zealand. The paired-box (PAX) genes encode a family of nine well-characterized paired-box transcription factors, with important roles in development and disease. Although PAX genes are primarily expressed in the embryo, constitutive expression promotes tissue hyperplasia. Rare tumor-specific mutations of PAX genes implicate an oncogenic role, and persistent PAX expression characterizes several tumors. Yet, a cancer-wide analysis of PAX gene expression to investigate a general role for PAX genes has not been performed. We analysed the pattern and requirement for PAX gene expression in a panel of common cancer cell lines. Very frequent PAX gene expression was identified in tumor cell lines, including lymphoma, breast, ovarian, lung, and colon cancer. In addition, the PAX2 gene was frequently expressed in a panel of 406 common primary tumor tissues. Apoptosis was rapidly induced in ovarian and bladder cancer cell lines following RNA interference to silence PAX2 expression, despite concomitant TP53 and/or HRAS mutations. These data suggest that PAX genes are frequently expressed in cancer, and that endogenous PAX gene expression is required for the growth and survival of cancer cells. PMID: 12970747 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Anticancer Drugs. 2002 Sep;13(8):881-9. Selective chemosensitization of rhabdomyosarcoma cell lines following wild-type p53 adenoviral transduction. Shetty S, Taylor AC, Harris LC. Department of Molecular Pharmacology, St Jude Children's Research Hospital, Memphis, TN 38105, USA. Rhabdomyosarcoma (RMS) cell lines were transduced with an adenoviral vector containing the wild-type p53 (wtp53) cDNA (Ad-p53) and then exposed to four cytotoxic agents: actinomycin D, vincristine, 5-fluorouracil and bleomycin. Potentiation of cytotoxicity following wild-type p53 expression varied from 0- to 20-fold for different drugs and between cell lines. It appeared that alveolar RMS cells (n = 2) were more susceptible to p53-mediated chemosensitization than embryonal RMS cells (n = 3), although this was independent of pax3-FKHR expression. Overall, cells that were most chemosensitive prior to Ad-p53 exposure were those that were most susceptible to p53 potentiation of cytotoxicity. The different results obtained with these RMS cell lines does not appear to be related to expression of pax3-FKHR, p21, Bax or Bcl-2 but may in part be due to differential regulation of p53 target genes, such as MDM2. In conclusion, exogenous wild-type expression selectively chemosensitizes RMS cells to cytotoxic agents. However, expression of transcriptionally active wtp53 does not predict a chemosensitive phenotype. PMID: 12394275 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: JAMA. 2002 May 15;287(19):2487-8. Causes sought for neural tube defects in infants of diabetic pregnant women. Friedrich MJ. Publication Types: News PMID: 12020316 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Genes Dev. 2002 Mar 15;16(6):676-80. Rescue of neural tube defects in Pax-3-deficient embryos by p53 loss of function: implications for Pax-3- dependent development and tumorigenesis. Pani L, Horal M, Loeken MR. Section on Cellular and Molecular Physiology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, USA. Pax-3 is a transcription factor that is expressed in the neural tube, neural crest, and dermomyotome. We previously showed that apoptosis is associated with neural tube defects (NTDs) in Pax-3-deficient Splotch (Sp/Sp) embryos. Here we show that p53 deficiency, caused by germ-line mutation or by pifithrin-alpha, an inhibitor of p53-dependent apoptosis, rescues not only apoptosis, but also NTDs, in Sp/Sp embryos. Pax-3 deficiency had no effect on p53 mRNA, but increased p53 protein levels. These results suggest that Pax-3 regulates neural tube closure by inhibiting p53-dependent apoptosis, rather than by inducing neural tube-specific gene expression. PMID: 11914272 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Genes Chromosomes Cancer. 1999 Dec;26(4):275-85. Genes, chromosomes, and rhabdomyosarcoma. Anderson J, Gordon A, Pritchard-Jones K, Shipley J. Unit of Molecular Hematology and Oncology, Institute of Child Health, London, United Kingdom. Rhabdomyosarcomas are a heterogeneous group of malignant tumors and are the most common soft-tissue sarcoma of childhood. Rhabdomyosarcomas resemble developing skeletal muscle, notably in their expression of the MRF family of transcription factors and the PAX3 and PAX7 genes. These PAX genes are also involved through specific translocations, t(2;13)(q35;q14) and variant t(1;13)(p36;q14) in the alveolar subtype, which result in PAX3-FKHR and PAX7-FKHR fusion genes, respectively. The fusion genes are thought critically to affect downstream targets of PAX3 and PAX7 or possibly have novel targets. Similar downstream changes may also be involved in embryonal and fusion gene negative cases. Genomic amplification of such genes as MYCN, MDM2, CDK4, and PAX7-FKHR is a feature mainly of the alveolar subtype, while specific chromosomal gains, including chromosomes 2, 8, 12, and 13, are associated with the embryonal subtype. Loss of alleles and imprinting at 11p15.5 and disruption of genes such as IGF2, ATR, PTC, P16, and TP53 have also been implicated in rhabdomyosarcoma development. Whereas there is now a realistic possibility of cure in the majority of cases, there remains a subset that is resistant to multimodality therapy, including high-dose chemotherapy. Characterization of the defining molecular features of tumors that are likely to behave aggressively represents a particular challenge. Current research is leading toward a better understanding of rhabdomyosarcoma tumorigenesis, which may ultimately result in novel therapeutic strategies that increase the overall cure. Genes Chromosomes Cancer 26:275-285, 1999. Copyright 1999 Wiley-Liss, Inc. Publication Types: Review Review, Tutorial PMID: 10534762 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Gene. 1998 Nov 26;223(1-2):303-8. A conserved TN8TCCT motif in the octapeptide-encoding region of Pax genes which has the potential to direct cytosine methylation. Ziman MR, Kay PH. Department of Pathology, The University of Western Australia, Nedlands, Western Australia, 6009, Australia. Our previous findings have shown that the developmental genes Pax7 and Pax3 are differentially methylated; the gene region that encodes the paired domain is hypomethylated, whereas the region that encodes the homeodomain is hypermethylated. For this reason, the known DNA sequence between the paired and homeoboxes was analysed for the presence of a conserved DNA motif to which a modifying protein could bind in order to direct the methylation or demethylation of surrounding gene sequences. The octapeptide-encoding region was found to contain several nucleotides that were highly conserved throughout the Pax gene family from phylogenetically distant species. The most conserved nucleotides are thought to comprise a motif TN8TCCT where N8=any combination of eight nucleotides. A conserved octapeptide-like-encoding sequence containing the TN8TCCT motif was also found in non-Pax genes of higher eukaryotes and in the non-coding strand of plants. Moreover, differential methylation seems to be associated with the presence of the TN8TCCT motif in p53 and the human oestrogen receptor genes. The presence of the TN8TCCT motif within an octapeptide-like-encoding sequence in human T-cell leukaemia virus type 1 might suggest that the putative recognition motif may have been introduced into various host genomes via some form of retroviral agent. PMID: 9858754 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Anticancer Res. 1998 Mar-Apr;18(2A):849-53. The mutation status of PAX3 and p53 genes in medulloblastoma. Wang W, Kumar P, Wang W, Whalley J, Schwarz M, Malone G, Haworth A, Kumar S. Department of Biological Sciences, Manchester Metropolitan University, UK. The p53 and PAX3 genes were examined by PCR, SSCP and DNA sequencing methods in 50 and 58 paraffinembedded medullablastoma tissues, respectively. Four novel mutations were identified among these samples in exon 5 of the p53 gene. Two tumours showed a G to A transition. One heterozygous mutation was located on codon 158 which changed the encoded amino acid from Arg (CGC) to His (CAC). Another was located on codon 174 and replaced AGG (Arg) with AAG (Lys). There was a single base deletion of guanine located on codon 160 in another two samples, causing a frameshift. This is the first study of mutation status of PAX gene in medulloblastoma wherein only one polymorphism was identified in the gene. The polymorphism changed codon 43 from GGC to GGT but both encoded glycine. PMID: 9615731 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Clin Cancer Res. 1998 Jan;4(1):145-52. Selective sensitization to DNA-damaging agents in a human rhabdomyosarcoma cell line with inducible wild-type p53 overexpression. Gibson AA, Harwood FG, Tillman DM, Houghton JA. Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. Drug-induced cytotoxicity or apoptosis may be influenced by the expression of the p53 tumor suppressor gene and by the specific oncogene expressed, which may dictate the threshold at which a cytotoxic response may by induced. The objective of the study was to elucidate how DNA-damaging agents with different mechanisms of action were sensitized in the context of expression of the Pax3/FKHR fusion protein, a transformation event unique to alveolar rhabdomyosarcomas (ARMSs), and wild-type p53 (wtp53). A wtp53 cDNA was subcloned into the pGRE5-2/EBV vector with dexamethasone-inducible overexpression and transfected into Rh30 ARMS cells that express Pax3/FKHR and a mutant p53 phenotype. Following dexamethasone induction of wtp53 overexpression in a derived clone (Cl.#27), growth was slowed, and cells accumulated in G1. Functional wtp53 activity was demonstrated by selective transactivation of p50-2, a wtp53 chloramphenicol acetyltransferase reporter construct, and by up-regulated expression of endogenous p21Waf1. Data demonstrated p53-dependent sensitization (> or = 4-fold) to bleomycin, actinomycin D, and 5-fluorouracil and considerably less p53-dependence (< or = 2-fold) for doxorubicin, topotecan, etoposide, and cisplatin in Cl.#27 compared to an equivalent clone containing the pGRE5-EBV vector alone (VC#3). Data demonstrate that ARMS cells show a selective sensitization to DNA-damaging agents when wtp53 is overexpressed. The cytotoxic activity of agents that are not potentiated substantially must, therefore, depend upon p53-independent factors that relate to the mechanism of drug action. PMID: 9516963 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------