2D-Difference Gel Electrophoretic Proteomic Analysis of a Cell Culture Model of Alveolar Rhabdomyosarcoma

Pressey, Joseph G.; Pressey, Christine S.; Robinson, Gloria; Herring, Richie; Wilson, Landon; Kelly, David R.; Kim, Helen

doi:10.1021/pr1008493

Cited by 15 publications

(8 citation statements)

References 38 publications

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“…RNA was subjected to reverse transcription and the gene of interest and a duplexed 18S rRNA internal control were measured by qRT-PCR on an ABI-Prism 7900HT Sequence Detection System. Quantification of gene expression was calculated by the ΔΔCt method [18,19]. Calibrator samples for each assay were either unfractionated RD or RH30 cells.…”

Section: Methodsmentioning

confidence: 99%

CD133 marks a myogenically primitive subpopulation in rhabdomyosarcoma cell lines that are relatively chemoresistant but sensitive to mutant HSV

Pressey¹,

Haas²,

Pressey³

et al. 2012

Pediatric Blood & Cancer

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Background Rhabdomyosarcoma (RMS) is characterized by features of skeletal muscle and is comprised of two major histological subtypes, embryonal (E-RMS) and alveolar (A-RMS). Subsets of each RMS subtype demonstrate resistance to multimodal therapy leading to treatment failure. Cancer stem cells or cancer-initiating cells (CIC) represent a theorized population of cells that give rise to tumors and are responsible for treatment resistance. Procedure We investigated the ability of CD133, a putative CIC marker, to distinguish a chemoresistant, myogenically primitive population in alveolar (RH30) and embryonal (RD) RMS cell lines. We tested CD133+/- cells for sensitivity to engineered herpes simplex virus (oHSV) Results Relative to CD133- cells, CD133+ A-RMS and E-RMS cells demonstrate an enhanced colony-forming ability, are less differentiated myogenically, and are more resistant to cytotoxic chemotherapy but equally sensitive to oHSV oncolysis. Compared to CD133- RD cells, CD133+ cells express relatively high levels of genes typically expressed in skeletal muscle progenitor satellite cells including PAX7, c-MET, and the GLI effectors of the hedgehog signaling pathway. In contrast, CD133+ RH30 cells were not associated with enhanced expression of satellite cell markers or Hh targets. Conclusions Our findings demonstrate that CD133+ cells from A-RMS and E-RMS cell lines are characterized by a myogenically primitive phenotype. These cells have the capacity to form colonies in vitro and are more resistant to chemotherapy than CD133- cells. CD133 expression may denote a subset of RMS cells with an important role in tumorigenesis and treatment failure. These resistant cells may be effectively targeted by oHSV therapy.

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Section: Methodsmentioning

confidence: 99%

CD133 marks a myogenically primitive subpopulation in rhabdomyosarcoma cell lines that are relatively chemoresistant but sensitive to mutant HSV

Pressey¹,

Haas²,

Pressey³

et al. 2012

Pediatric Blood & Cancer

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“…Mass spectrometry analysis was conducted at the Targeted Metabolomics and Proteomics Laboratory at the University of Alabama at Birmingham as described previously (25). Briefly, protein bands from SDS-PAGE gels were excised and The tandem mass spectrometry data thus obtained were processed to provide protein identification using an in-house MASCOT search engine (Matrix Science) with the M. smegmatis NCBInr protein database.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Two Adenosine Phosphorylase Activities in Mycobacterium smegmatis

Buckoreelall

Wilson

Parker

2011

Journal of Bacteriology

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Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine ؉ phosphate 7 adenine ؉ alpha-D-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent K m (K m app ) of 98 ؎ 6 M (with Ado) and a native molecular mass of 125 ؎ 7 kDa. The second Ado cleavage activity was identified as 5-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.

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“…Other in vitro models that have been used involve the introduction of the PAX3/7-FOXO1 fusion proteins into both myogenic and non-myogenic cell lines including fibroblast cell lines, ERMS cell lines, MEFs, mesenchymal stem cells (MSC), normal or immortalized human or mouse myogenic cells and even an osteosarcoma cell line [5,81,82,91,114-119]. Given that the cell of origin for ARMS has yet to be identified, perhaps this variation in model cell lines is prudent.…”

Section: Reviewmentioning

confidence: 99%

Alveolar rhabdomyosarcoma – The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis

Marshall

Grosveld

2012

Skeletal Muscle

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Rhabdomyosarcoma is a soft tissue sarcoma arising from cells of a mesenchymal or skeletal muscle lineage. Alveolar rhabdomyosarcoma (ARMS) is more aggressive than the more common embryonal (ERMS) subtype. ARMS is more prone to metastasis and carries a poorer prognosis. In contrast to ERMS, the majority of ARMS tumors carry one of several characteristic chromosomal translocations, such as t(2;13)(q35;q14), which results in the expression of a PAX3-FOXO1 fusion transcription factor. In this review we discuss the genes that cooperate with PAX3-FOXO1, as well as the target genes of the fusion transcription factor that contribute to various aspects of ARMS tumorigenesis. The characterization of these pathways will lead to a better understanding of ARMS tumorigenesis and will allow the design of novel targeted therapies that will lead to better treatment for this aggressive pediatric tumor.

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2D-Difference Gel Electrophoretic Proteomic Analysis of a Cell Culture Model of Alveolar Rhabdomyosarcoma

Cited by 15 publications

References 38 publications

CD133 marks a myogenically primitive subpopulation in rhabdomyosarcoma cell lines that are relatively chemoresistant but sensitive to mutant HSV

CD133 marks a myogenically primitive subpopulation in rhabdomyosarcoma cell lines that are relatively chemoresistant but sensitive to mutant HSV

Identification and Characterization of Two Adenosine Phosphorylase Activities in Mycobacterium smegmatis

Alveolar rhabdomyosarcoma – The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis

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