This selection from the NCCN Guidelines for Adolescent and Young Adult (AYA) Oncology focuses on treatment and management considerations for AYA patients with cancer. Compared with older adults with cancer, AYA patients have unique needs regarding treatment, fertility counseling, psychosocial and behavioral issues, and supportive care services. The complete version of the NCCN Guidelines for AYA Oncology addresses additional aspects of caring for AYA patients, including risk factors, screening, diagnosis, and survivorship.
Expression of the src homology 3 (SH3) domain-containing expressed in tumorigenic astrocytes (SETA) gene is associated with the tumorigenic state in astrocytes. SETA encodes a variety of adapter proteins containing either one or two SH3 domains, as suggested by the sequence heterogeneity of isolated cDNAs. Using both SH3 domains in a yeast two-hybrid screen of a glial progenitor cell cDNA library, we isolated the rat homolog of the ALG-2-interacting protein 1 or ALG-2-interacting protein X (AIP1/Alix). In vitro confrontation experiments showed that the SH3-N domain of SETA interacted with the proline-rich C terminus of AIP1. In co-immunoprecipitation experiments, SETA and AIP1 interacted and could form a complex with apoptosis-linked gene 2 protein. Endogenous SETA and AIP1 proteins showed similar patterns of staining in primary rat astrocytes. Misexpression of a variety of SETA protein isoforms in these astrocytes revealed that they localized to the actin cytoskeleton. Furthermore, SETA proteins containing the SH3-N domain were able to sensitize astrocytes to apoptosis induced by UV irradiation. Expression of the isolated SH3-N domain had the greatest effect in these experiments, indicating that interference in the interaction between endogenous SETA and AIP1 sensitizes astrocytes to apoptosis in response to DNA damage. Expression of the src homology 3 (SH3) domain-containing expressed in tumorigenic astrocytes (SETA) gene is associated with the tumorigenic state in astrocytes. SETA encodes a variety of adapter proteins containing either one or two SH3 domains, as suggested by the sequence heterogeneity of isolated cDNAs. Using both SH3 domains in a yeast two-hybrid screen of a glial progenitor cell cDNA library, we isolated the rat homolog of the ALG-2-interacting protein 1 or ALG-2-interacting protein X (AIP1/Alix). In vitro confrontation experiments showed that the SH3-N domain of SETA interacted with the proline-rich C terminus of AIP1. In co-immunoprecipitation experiments, SETA and AIP1 interacted and could form a complex with apoptosis-linked gene 2 protein. Endogenous SETA and AIP1 proteins showed similar patterns of staining in primary rat astrocytes. Misexpression of a variety of SETA protein isoforms in these astrocytes revealed that they localized to the actin cytoskeleton. Furthermore, SETA proteins containing the SH3-N domain were able to sensitize astrocytes to apoptosis induced by UV irradiation. Expression of the isolated SH3-N domain had the greatest effect in these experiments, indicating that interference in the interaction between endogenous SETA and AIP1 sensitizes astrocytes to apoptosis in response to DNA damage. srchomology 3 apoptosis-linked gene 2 ALG-2-interacting protein 1 ALG-2-interacting protein X Dulbecco's modified Eagle's medium glutathione S-transferase kilobase SH3 domain-containing expressed in tumorigenic astrocytes TdT dUTP nick end labeling The src homology 3 (SH3)1 domain-containing expressed in tumorigenic astrocytes (SETA) gene was isolated from differentiating glial cells and implicated in primary brain tumors on the basis of its expression in malignant astrocytes in culture and gliomas in the adult brain in vivo (1.Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.-J.S. Cavenee W.K. Neuro-Oncology. 2000; 2: 6-15Crossref PubMed Google Scholar). While SETA mRNA is expressed in the developing rodent brain at high levels, it is barely detectable in the adult rat or mouse cortex or in normal human brain. However, approximately half of all experimentally induced rat gliomas as well as human astrocytomas of grade II, III, and IV express the gene, and it is also found in oligodendrogliomas and oligoastrocytomas (1.Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.-J.S. Cavenee W.K. Neuro-Oncology. 2000; 2: 6-15Crossref PubMed Google Scholar). Furthermore, expression of SETA in a culture model of astrocytoma progression based on astrocytes from p53 knockout mice (2.Bogler O. Huang H.J. Cavenee W.K. Cancer Res. 1995; 55: 2746-2751PubMed Google Scholar, 3.Bogler O. Nagane M. Gillis J. Huang S.H.-J. Cavenee W.K. Cell Growth Differ. 1999; 10: 73-86PubMed Google Scholar) was closely associated with the ability of these cells to form tumors when reintroduced into animals (1.Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.-J.S. Cavenee W.K. Neuro-Oncology. 2000; 2: 6-15Crossref PubMed Google Scholar). In this model system, SETA shared expression patterns with established glioma-associated genes including epidermal growth factor receptor, platelet-derived growth factor receptors α and β, vascular endothelial cell growth factor, and protein kinase C-δ (3.Bogler O. Nagane M. Gillis J. Huang S.H.-J. Cavenee W.K. Cell Growth Differ. 1999; 10: 73-86PubMed Google Scholar). These expression data suggest that the re-expression of the SETA protein in astrocytes in the mature central nervous system contributes to their malignant transformation and progression, prompting an investigation of its mode of action at the molecular level. The SETA gene encodes proteins that contain SH3 domains, which are involved in high affinity, specific protein-protein interactions. While these domains are found in proteins with enzymatic function, such as Src kinase, they are also common in adapter molecules whose function is to promote the interaction of members of signal transduction pathways. SETA appears to belong in this group and to be a member of a new subfamily of adapter molecules that includes the CD2AP and CMS proteins (4.Dustin M.L. Olszowy M.W. Holdorf A.D. Li J. Bromley S. Desai N. Widder P. Rosenberger F. van der Merwe P.A. Allen P.M. Shaw A.S. Cell. 1998; 94: 667-677Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 5.Kirsch K.H. Georgescu M.-M. Ishimura S. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6211-6216Crossref PubMed Scopus (131) Google Scholar). In addition to SH3 domains, these proteins appear to have coiled-coil motifs at their C termini and two PXXP motifs, which are themselves the cognates of SH3 domains. Therefore, they appear to have at least three modalities of binding to other proteins, suggesting involvement in a complex series of protein-protein interactions. While CD2AP has been shown to interact with the CD2 molecule in T-cells (4.Dustin M.L. Olszowy M.W. Holdorf A.D. Li J. Bromley S. Desai N. Widder P. Rosenberger F. van der Merwe P.A. Allen P.M. Shaw A.S. Cell. 1998; 94: 667-677Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), CMS has been shown to interact with actin, p130 cas, the p85 subunit of phosphatidylinositol 3-kinase,src family kinases, and Grb2 (5.Kirsch K.H. Georgescu M.-M. Ishimura S. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6211-6216Crossref PubMed Scopus (131) Google Scholar), suggesting that this family of proteins may play a role in cell architecture and mitogenic signaling. As a first step toward understanding the function of SETA, we have taken the direct approach of isolating a binding partner with a yeast two-hybrid library screen. The protein we isolated is the rat homolog of the mouse gene, apoptosis-linked gene 2 (ALG-2)-interacting protein 1 or ALG-2-interacting protein X (AIP1/Alix), which was recently isolated by two groups performing yeast two-hybrid screens with the ALG-2 (6.Missotten M. Nichols A. Rieger K. Sadoul R. Cell Death Differ. 1999; 6: 124-129Crossref PubMed Scopus (212) Google Scholar, 7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). In vitro confrontation and co-immunoprecipitation from transiently transfected cells demonstrated that the SH3-N domain of SETA interacts with the proline-rich C terminus of AIP1 and that SETA, AIP1, and ALG-2 can form a complex. ALG-2 was originally isolated in a "death trap" screen in T-cells, and an antisense ALG-2 cDNA promoted survival after apoptosis had been induced by a variety of stimuli (8.Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (456) Google Scholar). Conversely, when ALG-2 was overexpressed in fibroblasts it sensitized them to cell death. Therefore, it has been suggested that the 22-kDa calcium-binding protein ALG-2 is a necessary component of the apoptotic machinery (8.Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (456) Google Scholar). AIP1 is a 105-kDa protein with a proline-rich C terminus that has 10 PXXP sequence motifs of the kind that bind to SH3 domains (7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). While the mechanism of action of AIP1 alone or in conjunction with ALG-2 is not understood, it is clear that perturbing the levels of these proteins in a variety of cells alters their response to apoptotic stimuli without affecting the background level of apoptosis (7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 8.Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (456) Google Scholar). In this paper, we show that these genes are expressed in astrocytes and glioma cells, which also express endogenous SETA proteins. Furthermore, introducing SETA proteins capable of binding to AIP1 sensitized astrocytes to UV light-induced cell death, while control cells or those expressing a part of the SETA protein that does not bind AIP1 had no effect. These data suggest that SETA, AIP1, and ALG-2 represent a new set of proteins with a role in modulating apoptosis in astrocytes and with a potential role in the formation of gliomas. The SETA SH3-NC bait construct insert was generated by polymerase chain reaction from a cDNA template using proofreading DeepVent DNA polymerase (New England Biolabs), and the absence of mutations was confirmed by sequencing. It included amino acids 24–258 (ERQRR … LPSDF; see Fig.2 A) according to the numbering of the longer SETA isoform described in Ref. 1.Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.-J.S. Cavenee W.K. Neuro-Oncology. 2000; 2: 6-15Crossref PubMed Google Scholar and GenBankTMAF131867. This insert was introduced into the pBD-GAL4 phagemid vector (Stratagene) and was used to screen a CG4 rat glial progenitor cell library (1.Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.-J.S. Cavenee W.K. Neuro-Oncology. 2000; 2: 6-15Crossref PubMed Google Scholar) previously constructed in HybriZAP and rescued into the pAD-GAL4 plasmid form (Stratagene). Two rounds of yeast colony screening for the ability to grow in the absence of histidine and for a positive β-galactosidase reaction were performed. Isolated plasmids were sequenced, and the sequences were compared with gene data bases. SETA SH3 cDNAs (see legend to Fig. 2 A for details) were cloned in frame into pGEX-KG, and glutathione S-transferase (GST) fusion proteins were generated and purified on glutathione-Sepharose 4B according to the manufacturer's instructions (Amersham Pharmacia Biotech). The plasmid encoding the ALG-2 GST fusion protein (pGEX-ALG-2) as well as mouse AIP1 (pCDNA3-AIP1) were obtained from Luciano D'Adamio and have been described previously (7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). [3H]Leucine-labeled AIP1 and β-galactosidase were generated by coupled in vitrotranscription and translation (TNT; Promega) and confronted with an excess of SETA or ALG-2 GST fusion proteins in 20 mm Tris, pH 7, 100 mm NaCl, 1 mm EGTA, 0.96 mm CaCl2, and 0.1% Nonidet P-40, which contains 5 μm free calcium. Complexes were collected using glutathione-Sepharose 4B, washed in several changes of buffer, and separated on an SDS-polyacrylamide gel, which was prepared for fluorography and exposed to film. SETA cDNAs (see legend to Fig. 2 A for details) were cloned in frame into pcDNA4-Xpress/His (SH3-N, SH3-C, and coiled coil (SETA NCcc)) or pcDNA6-V5/His (SETA SH3-NC, -N, and -NC), which provide the X-press or V5 epitope tag, respectively (Invitrogen). These plasmids were then co-transfected into 293 cells with murine AIP1-FLAG, obtained from Luciano D'Adamio, by the calcium phosphate method. Two days after transfection, cell lysates were prepared in radioimmune precipitation assay buffer, and immunoprecipitates were generated using monoclonal antibodies directed against the V5, Xpress (Invitrogen), or FLAG (Sigma) epitopes and collected on protein A-agarose (Roche Molecular Biochemicals). Precipitates were also generated using bacterially made ALG-2 GST fusion protein, as described above. Precipitates were Western blotted to polyvinylidene difluoride membrane; exposed to V5, Xpress, FLAG, or anti-SETA antibodies; and detected by chemiluminescence (Bio-Rad). Poly(A)+ mRNA was isolated using poly(dT) beads (Oligotex, Qiagen), and known amounts were subjected to Northern blotting. Membranes were hybridized with random primed 32P-labeled (Prime-It; Stratagene) probes derived from murine AIP1 and ALG-2 cDNAs provided by Luciano D'Adamio. Initial attempts at obtaining astrocytes expressing SETA proteins encoded by pCDNA vectors resulted in the isolation of clones that showed low levels and percentages of expression. Therefore, pcDNA4-Xpress/His (SETA NCcc)- and pcDNA6-V5/His (SETA SH3-NC, -N, and -C)-derived inserts including the vector-derived epitope tags were recloned into a retroviral vector, 1726/zeo. This retrovirus is a modification of 1726, itself a modification of LNL6 encoding theneo gene, an encephalomyocarditis virus-derived internal ribosome entry site (9.Sugimoto Y. Aksentijevich I. Gottesman M.M. Pastan I. Bio/Technology. 1994; 12: 694-698Crossref PubMed Scopus (74) Google Scholar), and the lacZ gene. TheSpeI (870)–XbaI (7093) fragment of 1726 was isolated using a partial XbaI cut and inserted intoSpeI XbaI double-digested LNCX (another LNL6 derivative modified by, and a kind gift from, Dr. Bob Navaiaux), which lacked the EcoRI site at position 1 of LNL6. After removal of the neo gene by EcoRI digestion and religation, this left a single EcoRI site immediately downstream of the packaging signal for the insertion of genes of interest. The lacZ gene was removed and replaced by a polymerase chain reaction-generated zeo gene, derived from pSV40/Zeo (Invitrogen), inserted in frame with the internal ribosome entry site using NcoI and XhoI restriction enzymes to generate 1726/zeo. SETA cDNAs derived from pCDNA plasmids were inserted blunt into EcoRI-digested and blunted 1726/zeo, and their orientation was determined by sequencing. Pseudotyped retrovirus was generated by transient co-transfection of 293GP cells with an expression plasmid encoding vesicular stomatitis virus G protein (10.Bartz S.R. Vodicka M.A. Methods. 1997; 12: 337-342Crossref PubMed Scopus (132) Google Scholar) and 1726/zeo constructs. Primary rat astrocytes were plated at a density of 50,000 cells in a 25-cm2 tissue culture flask and infected the following day with SETA-encoding retrovirus for 4 h in the presence of 8 μg/ml polybrene. Two days later, cells were passaged and replated in a 75-cm2 tissue culture flask and exposed to 300 μg/ml zeocin (Invitrogen). Cultures were maintained until uninfected control cultures contained no viable cells, typically 7–10 days, at which point they were harvested and replated at nominal passage 1. Cells were maintained in 300 μg/ml zeocin at all times and analyzed for expression by Western blot by chemiluminescence detection (Bio-Rad) and immunohistochemistry using standard protocols. Polyclonal anti-SETA antibodies are as described (1.Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.-J.S. Cavenee W.K. Neuro-Oncology. 2000; 2: 6-15Crossref PubMed Google Scholar). Polyclonal anti-AIP1 antibodies were obtained from Pasquale Vito, and are as described previously (7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Immunohistochemistry was performed on fixed cells grown on coverslips and with ALEXA (Molecular Probes, Inc., Eugene, OR) or fluorescein isothiocyanate-conjugated (Southern Biotechnology Associates) secondary antibodies. Cells were counterstained with phalloidin-rhodamine or anti-paxillin antibodies (Sigma). For analysis of apoptosis, cultures of astrocytes growing on coverslips in 6-cm tissue culture dishes with 5 ml of medium were exposed to 5 mJ/cm2 of UV irradiation in a GS Genelinker UV chamber (Bio-Rad). Coverslips were removed 24 h later and prepared for analysis with annexin V (Pharmingen Becton-Dickinson) or TUNEL (Roche Biochemicals) according to the manufacturer's instructions. The SETA gene encodes an adapter molecule with SH3 domains, suggesting that it functions by binding with high affinity and specificity to other proteins. Therefore, a yeast two-hybrid cDNA library screen was performed to isolate potential binding partners of SETA. A bait construct encoding the two SH3 domains of SETA and the intervening sequence (for details, see "Experimental Procedures"), fused in frame to the DNA binding domain of the GAL4, was used to screen a CG4 glial progenitor cell line cDNA library. SETA was originally isolated from these cells (1.Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.-J.S. Cavenee W.K. Neuro-Oncology. 2000; 2: 6-15Crossref PubMed Google Scholar), making it likely that physiologically relevant binding partners would be represented. Analysis of only 250,000 co-transfectants led to the isolation of eight positive clones after two rounds of screening. Sequence analysis revealed that they represented two overlapping cDNAs for the rat homolog of the mouse AIP1/Alix gene (Fig.1), which had been previously isolated as a binding partner of ALG-2 (6.Missotten M. Nichols A. Rieger K. Sadoul R. Cell Death Differ. 1999; 6: 124-129Crossref PubMed Scopus (212) Google Scholar, 7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Interestingly, the partial rat cDNA clone isolated by us (Fig. 1) was only one amino acid shorter than the mouse cDNA isolated by yeast two-hybrid screening with ALG-2 (7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), suggesting that the same region of AIP1 interacts with both ALG-2 and SETA. Examination of the predicted protein sequence of the partial rat AIP1 cDNA revealed extensive sequence homology to the mouse AIP1 molecule (Fig. 1). Both predicted proteins encoded a proline-rich region, from amino acid 719 to the C terminus, in which close to 30% of the residues are proline, as compared with an overall frequency of 8% proline in the long form of AIP1 (6.Missotten M. Nichols A. Rieger K. Sadoul R. Cell Death Differ. 1999; 6: 124-129Crossref PubMed Scopus (212) Google Scholar, 7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Ten PXXP motifs, such as are found at the core of SH3 binding peptides (11.Mayer B.J. Eck M.J. Curr. Biol. 1995; 5: 364-367Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), can be found in this sequence, suggesting that it represents the region that interacts with SETA. Interestingly, although the rodent AIP1 proteins are highly homologous to the Xenopus geneXp95 (12.Che S. El-Hodiri H.M. Wu C.F. Nelman-Gonzalez M. Weil M.M. Etkin L.D. Clark R.B. Kuang J. J. Biol. Chem. 1999; 274: 5522-5531Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), the similarity is dramatically reduced in this proline-rich region (Fig. 1). To establish whether SETA and AIP1 can interact outside of the yeast two-hybrid system, in vitro confrontation experiments were performed. Bacterially generated GST fusion proteins, encoding either one or both of the SETA SH3 domains (Fig.2 A) or the ALG-2 molecule, were confronted with radiolabeled AIP1 made by coupled in vitro transcription and translation (Fig. 2 B) and then collected on glutathione-Sepharose. The SETA SH3-NC encoding GST fusion protein (Fig. 2 A), which contains the same region of SETA as was used in the bait construct employed in the yeast two-hybrid screen, interacted with AIP1 in vitro (Fig. 2 B). Furthermore, the isolated N-terminal SETA SH3 domain, fused to GST, was able to bind to mouse AIP1 in these experiments (Fig. 2 B). However, the C-terminal SH3 domain did not bind to AIP1 (Fig.2 A), which was not due to a general lack of function of this fusion protein, since it has been shown to bind to another novel SETA binding protein recently isolated in our laboratory (data not shown). As expected from previous studies, the ALG-2 protein was able to bind full-length AIP1 in this assay (6.Missotten M. Nichols A. Rieger K. Sadoul R. Cell Death Differ. 1999; 6: 124-129Crossref PubMed Scopus (212) Google Scholar, 7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). In contrast to the interaction between ALG-2 and AIP1, the SETA SH3-N protein was able to bind AIP1 in the absence of calcium, as would be expected from an SH3 domain-mediated interaction. Last, none of the GST fusion proteins interacted with radiolabeled β-galactosidase protein (Fig.2 B). The use of isolated SETA SH3 domains in these experiments provides the most precise definition of which regions of this protein are capable of interacting with AIP1, as functionality depends on correct folding of the SH3 domain, requiring the presence of their entire sequence. However, we were able to further define the region of AIP1 that bound to SETA by determining that neither the SH3-NC nor SH3-N proteins were able to interact with a truncated AIP1 protein, terminating at the internal EcoRI site at position 636 (Fig. 1), which lacks the proline-rich region of AIP1 (data not shown). Together, these data suggest that the interaction between SETA and AIP1 can occur outside of yeast and is mediated by SETA's N-terminal SH3 domain and AIP1's proline-rich C terminus. To test whether SETA and AIP1 also interact in cells, epitope-tagged proteins were transiently introduced into 293 cells, which can be transfected at high efficiency, and cell lysates were analyzed by immunoprecipitation and Western blotting (Fig. 2 C). A FLAG-tagged AIP1 cDNA was co-transfected with a C-terminally V5-tagged SETA SH3-NC expression construct, an N-terminally Xpress-tagged SETA NCcc construct or control lacZ constructs tagged with either epitope tag. Although in our hands the V5 epitope tag performed better in all experiments than the Xpress tag, the latter was chosen to tag SETA NCcc N-terminally in order to minimize the possibility of interfering with the structural integrity of the predicted C-terminal coiled-coil. Precipitates from the resultant lysates were collected with anti-V5, anti-Xpress, or anti-FLAG antibodies or with bacterially made ALG-2-GST. The resultant precipitates were then analyzed in Western blots probed with anti-V5, anti-Xpress, anti-SETA, or anti-FLAG antibodies. Antibodies against epitope tags were used in these experiments to specifically study transfected isoforms of SETA. In order to establish that immunoprecipitation with these antibodies recovered the transfected SETA proteins, lysates of 293 cells transfected with SETA SH3-NC and SETA NCcc were precipitated by anti-V5 or anti-Xpress antibodies, respectively, and immunoblotted with polyclonal anti-SETA antibodies. As shown in Fig. 2 C, lanes 1 and 2, bands of the expected size were obtained, demonstrating that these reagents were specifically recovering SETA proteins. Analysis of anti-Xpress-generated immunoprecipitates of cells transfected with AIP1-FLAG and SETA NCcc revealed a band of the size expected for the AIP1 protein (Fig. 2 C, lane 3), which was not present in lysates of cells transfected with the lacZ control construct (Fig. 2 C,lane 4). Bands common to both lanes 3 and 4 are anti-FLAG antibody cross-reacting proteins. To further support the suggestion that SETA and AIP1 interact in cells, lysates were immunoprecipitated with anti-FLAG antibodies recognizing AIP1. SETA SH3-NC or SETA NCcc was detected in these lysates with anti-V5 or anti-Xpress antibody, respectively (Fig.2 C, lanes 5 and 6). Therefore, complexes of AIP1 and SETA proteins could be recovered by immunoprecipitation with antibodies directed at either protein. To test whether SETA and AIP1 could bind to ALG-2 in one complex, we precipitated lysates with bacterially made GST-ALG-2 protein. In line with previous studies, GST-ALG-2 could precipitate AIP1-FLAG from cell lysates (Fig. 2 C, lane 7) (7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Furthermore, these lysates also contained SETA SH3-NC or SETA NCcc protein (Fig. 2 C, lanes 8 and9). Taken together, these data suggest that SETA and AIP1 can interact in cells. Furthermore, the detection of SETA proteins in precipitates made with GST-ALG-2, which does not interact directly with SETA, suggests that these two proteins can interact with AIP1 simultaneously. The observation that SETA, AIP1, and ALG-2 can interact raises the question of whether they are co-expressed in cells. SETA was originally identified as a gene associated with malignancy in astrocytes and is expressed in p53−/−astrocytes derived from p53 knockout mice capable of forming tumors as well as glioma-derived cell lines. Therefore, we analyzed these cells, as well as normal rat astrocytes expressing various SETA protein isoforms (see below), for AIP1 and ALG-2 mRNA expression. As shown in Fig. 3, all of the cells tested expressed AIP1 and ALG-2 mRNA. Two AIP1 mRNA species were observed, with one just below the 28 S at about 4 kb and one above the 28 S at approximately 7 kb, as described previously (7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). ALG-2 mRNA ran as a single band below the 18 S at approximately 1 kb (8.Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (456) Google Scholar). AIP1 and ALG-2 expression did not correlate with the ability of p53−/− astrocytes to form tumors, since similar levels of expression were found in cells grown in DMEM plus 10% fetal calf serum or in DMEM plus 20 ng/ml epidermal growth factor, which are capable of forming tumors, and DMEM plus 10% basic fibroblast growth factor, which are not (3.Bogler O. Nagane M. Gillis J. Huang S.H.-J. Cavenee W.K. Cell Growth Differ. 1999; 10: 73-86PubMed Google Scholar). Rat astrocytes with a normal p53 genotype also expressed both AIP1 and ALG-2 mRNA, regardless of whether they had been engineered to express SETA constructs or vector alone (see below). Last, both genes were also expressed in a rat glioma cell line, GV2C8, and two human glioma cell lines, LNZ308 and A172, all known to express SETA mRNA (1.Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.-J.S. Cavenee W.K. Neuro-Oncology. 2000; 2: 6-15Crossref PubMed Google Scholar). These data demonstrate that SETA, AIP1, and ALG-2 are co-expressed in a variety of cells relevant to the study of glial cell transformation and so have the opportunity to interact. To directly examine the question of whether the association of SETA with the known regulators of apoptosis ALG-2 and AIP1 allows it to modulate this process, primary rat astrocyte cell lines that expressed various SETA protein forms (shown in Fig.2 A) were established. Initial experiments in astrocytes demonstrated that the the plasmid expression vectors used in 293 cells (Fig. 2 C) did not achieve high enough efficiencies of transient transfection or maintain stable expression of SETA or control proteins in this cell type. Therefore, the epitope-tagged SETA cDNAs were engineered into a retroviral expression construct immediately downstream of the packaging signal and upstream of an internal ribosome entry site and the gene for zeocin resistance (for details, see "Experimental Procedures"). Infection of primary rat astrocytes with retroviruses generated from these constructs and selection with zeocin for 10 days resulted in populations of cells that showed greater than 98% expression of SETA proteins, which was stable over time, as determined by immunohistochemistry (data not shown). All further experiments were performed with these selected cell populations, thereby eliminating issues relating to clonal variation. Populations of astrocytes engineered with SETA SH3-NC in the antisense orientation or with vector alone were also isolated as controls. All of these populations expressed endogenous AIP1 and ALG-2 mRNA (Fig.3). Western blotting of zeocin-selected rat astrocyte populations with a polyclonal anti-SETA antibody revealed expression of endogenous SETA that increased with time in culture (Fig.4). Astrocytes cultured for fewer than five passages after the completion of selection showed variable, low levels of endogenous SETA, with bands near 60, 95, and 180 kDa (Fig. 4,lanes 1–6). At higher than 10 passages, the level of endogenous SETA was increased, as demonstrated by the inclusion of cell extract of high passage SETA NCcc-expressing astrocytes on the same Western blot as the low passage cells (Fig. 4,lane 7). This higher level of SETA expression was found in all cel
Abstract Ewing sarcoma (ES) is a malignant tumor of bone and soft tissue that most often occurs in adolescents and young adults. Despite an international coordinated approach, several nuances, discrepancies, and debates remain in defining the standard of care for treating ES. In this review, the authors leverage the expertise assembled by formation of the National Ewing Sarcoma Tumor Board, a multi‐institution, multidisciplinary virtual tumor board that meets monthly to discuss complicated and challenging cases of ES. This report is focused on select topics that apply to the management of patients with newly diagnosed ES. The specific topics covered include indications for bone marrow aspirate and biopsy for initial evaluation compared with fluorodeoxyglucose‐positron emission tomography, the role of interval compressed chemotherapy in patients aged 18 years and older, the role of adding ifosfamide/etoposide to vincristine/doxorubicin/cyclophosphamide for patients with metastatic disease, the data on and role of high‐dose chemotherapy with autologous stem cell transplantation, maintenance therapy, and whole‐lung irradiation. The data referenced are often limited to subgroup analyses and/or compiled from multiple sources. Although not intended to replace the clinical judgement of treating physicians, the guidelines are intended to provide clarity and recommendations for the upfront management of patients with ES. Plain Language Summary Ewing sarcoma is a malignant tumor of bone and soft tissue that most often occurs in adolescents and young adults. For this review, the authors used the experience of the National Ewing Sarcoma Tumor Board, a multi‐institution, multidisciplinary virtual tumor board that meets monthly to discuss complicated and challenging cases of Ewing sarcoma. Although not intended to replace the clinical judgement of treating physicians, the guidelines will focus on the development of consensus statements for the upfront management of patients with Ewing sarcoma.
10022 Background: Filgrastim (G-CSF) reduces the duration of severe neutropenia and incidence of febrile neutropenia following cytotoxic chemotherapy. Pegfilgrastim, a long acting form of G-CSF, has similar efficacy to G-CSF in adults, but studies in pediatrics are limited. We report our institutional experience with pegfilgrastim following dose intensive chemotherapy for solid tumors. Methods: Thirty-nine pediatric patients diagnosed with solid tumors between 1/1/07 and 11/14/07 who received dose intensive chemotherapy and pegfilgrastim therapy (100 mcg/kg; 6 mg maximum dose) were included in this retrospective review. Demographic data at diagnosis, treatment characteristics, frequency and duration of severe neutropenia (absolute neutrophil count < 0.2 ×109/L), and frequency of neutropenic fever were recorded. Results: The median age of patients evaluated was 12 yrs (range 0.17–23 years) and the median weight was 50 kg (range 4–107 kg); 13/39 (33×) weighed < 20 kg and 19/39 (49×) were less than 45 kg. Primary diagnoses included: osteosarcoma (6), Ewings sarcoma (5), rhabdomyosarcoma (9), soft tissue sarcoma (2), neuroblastoma (6), Hodgkin disease (6) and other solid tumors (5). In total, 141 chemotherapy courses with pegfilgrastim support were administered (median 4 courses per patient); no adverse events secondary to pegfilgrastim were recorded. Severe neutropenia occurred in 46× of courses. Overall, the median duration of severe neutropenia was 0 days (range 0–8 days). Febrile neutropenia occurred in 28× of courses. Of particular interest were 8 patients treated with interval-compressed (every 14 days) sarcoma chemotherapy. Of 51 courses administered, the median course duration was 15 days (range 14–28 days). Conclusions: Our study demonstrates that pegfilgrastim administration following dose intensive chemotherapy for solid tumors is safe and feasible in children, including those < 45 kg. The frequency and duration of severe neutropenia, as well as incidence of febrile neutropenia, were similar to G-CSF historic data. Pegfilgrastim is a suitable alternative to G-CSF therapy even in young children. No significant financial relationships to disclose.
Systemic corticosteroids are widely used for the treatment of acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma. Anecdotal case reports demonstrate bradycardia in patients receiving corticosteroids; however, a more in-depth analysis is lacking. This study aimed to describe the incidence, timing, and outcomes of bradycardia in children with ALL receiving corticosteroids during induction chemotherapy at our center from 2010 to 2016. A total of 153 children were included, with 150 (98%) demonstrating decreased heart rate following steroid administration with a median HR decrease of 23 beats per minute. Bradycardia ≤first percentile for age developed in 90 (59%) patients, with nadir occurring, on average, 7 doses into treatment, corresponding to 79 hours after initiation of therapy. No patient experienced adverse events related to bradycardia. Resolution of bradycardia at outpatient follow-up occurred in 62 of 71 (87%). Examination of nadir heart rate during subsequent hospitalizations in which steroids were not being administered did not demonstrate a significant incidence of bradycardia. Corticosteroid-induced bradycardia is common in children with ALL receiving induction chemotherapy. It was not associated with clinical adverse events and self-resolved without intervention. Therefore, further cardiac assessment may not be warranted in the presence of asymptomatic bradycardia suspected to be secondary to steroid administration.
Abstract Ewing sarcoma (ES) is a malignant tumor of bone and soft tissue that most often occurs in children, adolescents, and young adults. Debate and controversy remain in the management of relapsed/refractory ES (RR‐ES). The authors leveraged the expertise assembled by the National Ewing Sarcoma Tumor Board, a multidisciplinary virtual tumor board that meets monthly to discuss challenging cases of ES. In this review, they focus on select topics that apply to the management of patients with RR‐ES. The specific topics covered include the initial approach of such patients and discussion of the goals of care, the role of molecular testing, chemotherapy regimens and novel agents to consider, the role of maintenance therapy, and the use of high‐dose chemotherapy with autologous stem cell rescue. The data referenced are often limited to subgroup analyses and/or compiled from multiple sources. Although not intended to replace the clinical judgement of treating physicians, these guidelines are intended to support clinicians and provide some clarity and recommendations for the management of patients with RR‐ES. Plain Language Summary Ewing sarcoma (ES) is a bone and soft tissue cancer that most often occurs in teenagers and young adults. This article uses the experience of the National Ewing Sarcoma Tumor Board, a multi‐institution, multidisciplinary virtual tumor board that meets monthly to discuss challenging cases of ES and to address questions related to the treatment of patients with relapsed ES. Although not intended to replace the clinical judgement of treating physicians and limited by available data, these consensus recommendations will support clinicians who treat patients with this challenging malignancy, made even more difficult when it recurs.
Polycomb proteins are essential regulators of gene expression in stem cells and development. They function to reversibly repress gene transcription via posttranslational modification of histones and chromatin compaction. In many human cancers, genes that are repressed by polycomb in stem cells are subject to more stable silencing via DNA methylation of promoter CpG islands. Ewing sarcoma is an aggressive bone and soft-tissue tumor that is characterized by overexpression of polycomb proteins. This study investigates the DNA methylation status of polycomb target gene promoters in Ewing sarcoma tumors and cell lines and observes that the promoters of differentiation genes are frequent targets of CpG-island DNA methylation. In addition, the promoters of ion channel genes are highly differentially methylated in Ewing sarcoma compared with nonmalignant adult tissues. Ion channels regulate a variety of biologic processes, including proliferation, and dysfunction of these channels contributes to tumor pathogenesis. In particular, reduced expression of the voltage-gated Kv1.5 channel has been implicated in tumor progression. These data show that DNA methylation of the KCNA5 promoter contributes to stable epigenetic silencing of the Kv1.5 channel. This epigenetic repression is reversed by exposure to the DNA methylation inhibitor decitabine, which inhibits Ewing sarcoma cell proliferation through mechanisms that include restoration of the Kv1.5 channel function.This study demonstrates that promoters of ion channels are aberrantly methylated in Ewing sarcoma and that epigenetic silencing of KCNA5 contributes to tumor cell proliferation, thus providing further evidence of the importance of ion channel dysregulation to tumorigenesis.
