RNA helicase DDX3: a novel therapeutic target in Ewing sarcoma.

RNA helicase DDX3 has oncogenic activity in breast and lung cancers and is required for translation of complex mRNA transcripts, including those encoding key cell-cycle regulatory proteins. We sought to determine the expression and function of DDX3 in sarcoma cells, and to investigate the antitumor activity of a novel small molecule DDX3 inhibitor, RK-33. Utilizing various sarcoma cell lines, xenografts and human tissue microarrays, we measured DDX3 expression at the mRNA and protein levels, and evaluated cytotoxicity of RK-33 in sarcoma cell lines. To study the role of DDX3 in Ewing sarcoma, we generated stable DDX3-knockdown Ewing sarcoma cell lines using DDX3-specific small hairpin RNA (shRNA), and assessed oncogenic activity. DDX3-knockdown and RK-33-treated Ewing sarcoma cells were compared with wild-type cells using an isobaric mass-tag quantitative proteomics approach to identify target proteins impacted by DDX3 inhibition. Overall, we found high expression of DDX3 in numerous human sarcoma subtypes compared with non-malignant mesenchymal cells, and knockdown of DDX3 by RNA interference inhibited oncogenic activity in Ewing sarcoma cells. Treatment with RK-33 was preferentially cytotoxic to sarcoma cells, including chemotherapy-resistant Ewing sarcoma stem cells, while sparing non-malignant cells. Sensitivity to RK-33 correlated with DDX3 protein expression. Growth of human Ewing sarcoma xenografts expressing high DDX3 was inhibited by RK-33 treatment in mice, without overt toxicity. DDX3 inhibition altered the Ewing sarcoma cellular proteome, especially proteins involved in DNA replication, mRNA translation and proteasome function. These data support further investigation of the role of DDX3 in sarcomas, advancement of RK-33 to Ewing sarcoma clinical trials and development of RNA helicase inhibition as a novel anti-neoplastic strategy.

A dose-finding study of temsirolimus and liposomal doxorubicin for patients with recurrent and refractory bone and soft tissue sarcoma.

There are few effective therapies for high-risk sarcomas. Initial chemosensitivity is often followed by relapse. In vitro, mammalian target of rapamycin (mTOR) inhibition potentiates the efficacy of chemotherapy on resistant sarcoma cells. Although sarcoma trials using mTOR inhibitors have been disappointing, these drugs were used as maintenance. We conducted a Phase I/II clinical trial to test the ability of temsirolimus to potentiate the cytotoxic effect of liposomal doxorubicin and present here the dose-finding portion of this study. Adult and pediatric patients with recurrent or refractory sarcomas were treated with increasing doses of liposomal doxorubicin and temsirolimus using a continual reassessment method for escalation, targeting a dose-limiting toxicity rate of 20%. Blood samples were drawn before and after the first dose of temsirolimus in Cycles 1 and 2 for pharmacokinetic analysis. The maximally tolerated dose combination was liposomal doxorubicin 30 mg/m(2) monthly with temsirolimus 20 mg/m(2) weekly. Hematologic toxicity was common but manageable. Dose-limiting toxicities were primarily renal. Concurrent administration of liposomal doxorubicin resulted in increased exposure to sirolimus, the active metabolite of temsirolimus. Thus, the combination of liposomal doxorubicin and temsirolimus is safe for heavily pretreated sarcoma patients. Co-administration with liposomal doxorubicin did not alter temsirolimus pharmacokinetics, but increased exposure to its active metabolite.

Immunohistochemistry of affected tissue may guide cGVHD treatment decisions.

Chronic graft-vs-host disease (cGVHD) myositis is a rare complication of hematopoietic SCT, for which the pathogenesis and optimal therapy are unclear. We performed immunohistochemistry on muscle biopsies from pediatric cGVHD myositis and typical cases of autoimmune dermatomyositis and polymyositis. The immunostaining pattern of cGVHD myositis was distinct from that of typical cases of autoimmunity. There was a high proportion of CD20+ and CD68+ cells, and the best therapeutic response was achieved with rituximab (anti-CD20). These results suggest that cGVHD myositis may be mediated by different leukocytes than similar autoimmune diseases and that treatment may be optimized by targeting the specific cellular infiltrates identified in affected tissue.

Isoforms of Wilms' tumor suppressor gene (WT1) have distinct effects on mammary epithelial cells.

The role of WT1 (Wilm's tumor suppressor gene) in breast cancer is controversial, with evidence for both tumor-promoting and tumor-suppressing activities. In order to address this question, we expressed different WT1 isoforms in the mammary epithelial cell line H16N-2, which does not express endogenous WT1. Cells were stably transfected with either WT1 (-Ex5/-KTS) or WT1 (+Ex5/+KTS) under the control of the inducible metallothionein promoter. Induction of WT1 (-Ex5/-KTS) upregulated p21, causing a slowing of proliferation and a G2-phase cell cycle arrest. In artificial basement membrane, the WT1 (-Ex5/-KTS) isoform promoted the appearance of highly organized acinar cellular aggregates. In contrast, WT1 (+Ex5/+KTS) had no effect on p21 or proliferation, but rather caused an epithelial-mesenchymal transition and a redistribution of E-cadherin from the cell membrane to the cytoplasm. This isoform also causes the cellular aggregates growing in artificial basement membrane to appear significantly less organized than control cells. Thus, different WT1 isoforms have distinct effects in this cell line, suggesting that depending on the ratio of WT1 isoform expression in mammary epithelial cells, WT1 could function to either promote or suppress a transformed phenotype.

An isoform of the Wilms' tumor suppressor gene potentiates granulocytic differentiation.

WT1 is expressed in hematopoietic progenitor cells and in acute leukemia, but its role in normal and malignant hematopoiesis has not been clearly defined. Alternative splicing of the WT1 mRNA yields several protein isoforms with distinct DNA binding and transcriptional regulatory activities. In this study, we investigated the effect of the WT1 isoform lacking two alternatively spliced sequences (WT1 (-/-)) in 32D cl3 cells, a murine myeloid progenitor cell line. The expression of WT1 (-/-) accelerated the granulocyte-colony stimulating factor (G-CSF)-mediated differentiation of these cells, as judged by morphology and by the expression of differentiation-associated genes and cell surface antigens. WT1 (-/-) inhibited G1/S progression in G-CSF but not in interleukin-3, potentially accounting for its ability to accelerate differentiation. It is likely that dominant-negative mutants previously reported in leukemia patients participate in leukemogenesis by inhibiting this function of the wild-type protein.

Intensive timed sequential remission induction chemotherapy with high-dose cytarabine for childhood acute myeloid leukemia.

BACKGROUND: Timed sequential chemotherapy and high-dose cytarabine (cytosine arabinoside, Ara-C; HDAC) are both effective treatments for acute myeloid leukemia (AML). We review our institutional experience with timed sequential induction chemotherapy consisting of daunorubicin/Ara-C/-thioguanine (DAT) or idarubicin/Ara-C/-thioguanine (IAT) followed on day 14 by HDAC regardless of the degree of marrow aplasia for children with newly diagnosed AML. PROCEDURE: Children presenting with newly diagnosed AML were treated with induction chemotherapy consisting of idarubicin (12 mg/m/day on days 1-3 or daunorubicin at 45 mg/m(2)/day for the first five patients), Ara-C (100 mg/m(2)/day by continuous infusion on days 1-7), and thioguanine (100 mg/m(2)/day on days 1-7). HDAC (1 g/m(2)/dose every 12 hr for 10 doses) was administered beginning on day 14, regardless of the results of bone marrow examination. RESULTS: Thirteen children received timed sequential HDAC. Only one child received HDAC later than Day 18. Eleven of the children achieved a complete remission. All patients experienced grade 4 hematologic toxicity, and all had fever as well. There were 11 children with documented infections. Ten had grade 3 or 4 GI toxicity. One patient died of sepsis. CONCLUSIONS: HDAC administered as a part of timed sequential therapy yields an excellent remission induction rate with manageable toxicity.

Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation.

Cyclin D2 is a member of the D-type cyclins, implicated in cell cycle regulation, differentiation, and malignant transformation. It was noted previously that cyclin D2 is not expressed in the majority of breast cancer cell lines, whereas abundant expression was detected in finite life span human mammary epithelial cells. By reverse transcription-PCR and Western blot analysis, we extended this finding to primary breast carcinomas and show that the majority of these tumors lack expression of cyclin D2 mRNA (18 of 24) and protein (10 of 13). In contrast, both luminal and myoepithelial subpopulations of normal breast tissues expressed cyclin D2. Hypermethylation of the CpG island in the promoter was detected by methylation-specific PCR in nearly half of the breast cancers (49 of 106) and was associated with silencing of cyclin D2 gene expression. Promoter hypermethylation was also detected in ductal carcinoma in situ, suggesting that loss of cyclin D2 expression is an early event in tumorigenesis. Our results suggest that loss of cyclin D2 expression is associated with the evolution of breast cancer.

Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation.

We analyzed Wilms' tumor suppressor 1 (WT1) expression and its regulation by promoter methylation in a panel of normal breast epithelial samples and primary carcinomas. Contrary to previous reports, WT1 protein was strongly expressed in primary carcinomas (27 of 31 tumors) but not in normal breast epithelium (1 of 20 samples). Additionally, the WT1 promoter was methylated in 6 of 19 (32%) primary tumors, which nevertheless expressed WT1. The promoter is not methylated in normal epithelium. Thus, although tumor-specific methylation of WT1 is established in primary breast cancer at a low frequency, other transcriptional regulatory mechanisms appear to supercede its effects in these tumors. Our results demonstrate expression of WT1 in mammary neoplasia, and that WT1 may not have a tumor suppressor role in breast cancer.

Lymphoid malignancy as a presenting sign of ataxia-telangiectasia.

Ataxia-telangiectasia (AT) is an uncommon genetic disorder characterized by cerebellar ataxia, oculocutaneous telangiectasias, progressive immunodeficiency, and a predisposition to lymphoid malignancy. The genetic defect in AT predisposes not only to malignancy but also to severe toxicity from anti-neoplastic therapies. It is important to consider the diagnosis of AT in any child with a lymphoid malignancy at a younger than expected age, or who has a pre-existing ataxia, to anticipate unusually severe toxicities from the antineoplastic therapy, to avoid confusing the development of ataxia with toxicity from therapy, and to provide appropriate genetic counseling. We describe two children at a young age with a lymphoid malignancy diagnosed before the diagnosis of AT. One patient had severe toxicity from his chemotherapy, requiring truncation of the planned course of treatment. The other child was able to tolerate his entire planned course of therapy, but ataxia that was initially interpreted as toxicity from chemotherapy rather than as a sign of his AT developed. Lymphoid malignancy may be the presenting sign of AT. Making this diagnosis may influence therapy of the malignancy. The neurologic manifestations of the disease can be misinterpreted as toxicities of the chemotherapy, and diagnosis of AT allows appropriate genetic counseling for the family.

Regulation of granulopoiesis by transcription factors and cytokine signals.

The development of mature granulocytes from hematopoietic precursor cells is controlled by a myriad of transcription factors which regulate the expression of essential genes, including those encoding growth factors and their receptors, enzymes, adhesion molecules, and transcription factors themselves. In particular, C/EBPalpha, PU.1, CBF, and c-Myb have emerged as critical players during early granulopoiesis. These transcription factors interact with one another as well as other factors to regulate the expression of a variety of genes important in granulocytic lineage commitment. An important goal remains to understand in greater detail how these various factors act in concert with signals emanating from cytokine receptors to influence the various steps of maturation, from the pluripotent hematopoietic stem cell, to a committed myeloid progenitor, to myeloid precursors, and ultimately to mature granulocytes.

Oligo-/monoclonal gammopathy and hypergammaglobulinemia in ataxia-telangiectasia. A study of 90 patients.

We investigated the presence of hypergammaglobulinemia and oligo-/monoclonal gammopathy in 90 patients (from 80 families) with ataxia-telangiectasia ranging in age from 2 to 29 years. Of the 90 patients, 38.8% displayed hypergammaglobulinemia. An isolated increase in IgM was the most common finding (23.3%) followed by a simultaneous increase in IgM and IgG (8.8%), an isolated increase in IgA (3.3%), an elevated level of IgG (2.2%) and a concomitant increase in IgM and IgA (1.1%), respectively. Seven of the patients (8.1%) had oligo-/monoclonal gammopathy. The gammopathies included all major immunoglobulin isotypes. Chemotherapeutic intervention in 2 cases precipitated the emergence of new clones within a matter of weeks. Further investigation of oligo-/monoclonal gammopathies in these patients may lead to a clearer understanding of the clinical course and provide further insight into the underlying mechanisms of B-cell abnormalities in ataxia-telangiectasia.

Nerve growth factor induced stimulation of Ras requires Trk interaction with Shc but does not involve phosphoinositide 3-OH kinase.

The TrkA receptor protein tyrosine kinase is involved in signalling PC12 cell differentiation and cessation of cell division in response to nerve growth factor (NGF). To assess the importance of adaptor proteins and Ras in NGF control of phosphoinositide 3-OH kinase (PI 3-kinase), specific receptor mutations in Trk have been employed. We show that phosphorylation of tyrosine 490, but not 785, of Trk is essential for activation of both Ras and PI 3-kinase in vivo, correlating with tyrosine phosphorylation of Shc and binding of Shc to the adaptor Grb2 and the Ras exchange factor Sos. A mutant receptor that lacks Y490 and Y785, but contains an introduced YxxM motif which binds the regulatory domain of PI 3-kinase, is unable to activate Ras despite causing increased PI 3-kinase activity. This indicates clearly that activation of PI 3-kinase by itself is not sufficient to cause activation of Ras, arguing against a model in which PI 3-kinase acts upstream of Ras. The Shc site of Trk is thus crucial for the activation of Ras and PI 3-kinase.

Arthrography of the wrist. Assessment of the integrity of the ligaments in young asymptomatic adults.

Fifty-two asymptomatic adults who were between twenty and thirty-five years old had arthrography of the wrist with use of a single injection into the radiocarpal joint. The purpose of the study was to evaluate the integrity of the triangular fibrocartilage, the scapholunate ligament, and the lunotriquetral ligament. Contrast medium was injected under fluoroscopic guidance, and posteroanterior and lateral radiographs of the wrist were made after the subjects had performed exercises of the wrist. No patient who had a history of trauma to the wrist, pain in the wrist, or inflammatory arthritis was included in the study. All of the subjects had an examination of both upper extremities that included measurement of the active motion of the wrist with a goniometer, strength-testing with a Jamar dynamometer, ballottement and testing for impingement, and palpation for tenderness. Plain radiographs were evaluated, and the ulnar variance was recorded. The arthrograms revealed an abnormal communication of the contrast medium in fourteen wrists (27 per cent), and four of the fourteen had multiple areas of communication. The abnormal communication was through the triangular fibrocartilage alone in six wrists, the scapholunate ligament alone in two wrists, the lunotriquetral ligament alone in two wrists, and in more than one of these areas in four wrists. A positive arthrogram was associated with a greater positive ulnar variance. All of the subjects had symmetrical motion of the wrists and grip strength, and none of them had tenderness in the wrist. There were no complications related to the arthrography. Perforation of a ligament in the wrist is common in young asymptomatic adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiologic diagnosis of cystic fibrosis in adults and children.

RATIONALE AND OBJECTIVES: Most radiologists are familiar with the classic chest radiographic findings of cystic fibrosis (CF) when these occur in children. We hypothesized that given the same findings, a diagnosis of CF would be less likely to be considered in an adult than in a child. METHODS: We compiled 30 pediatric and 28 adult CF chest radiographs and obtained two independent readings on each by different general radiologists among the eight who volunteered to participate as they performed their daily clinical work. The cases were presented to the readers so that they did not know which radiographs were part of the study. The association between the correct diagnosis of CF and whether the patient was an adult or a child was assessed using odds ratios and logistic regression, so that Brasfield score, Schwachman-Kulczycki score, and the patient's sex could also be considered as predictive of correct diagnosis. RESULTS: In 67% of the pediatric cases, at least one of the radiologists considered CF as a possible diagnosis, whereas they considered CF a possibility in only 50% of the adults. Both radiologists suggested the correct diagnosis in 40% of pediatric cases and only 14% of adult cases (p < .05). CONCLUSION: Because the radiographic findings were similar in the two groups of patients according to severity groupings, we believe CF was less commonly considered in the adult patient because of the traditional belief that CF is a childhood disease.

A Trk nerve growth factor (NGF) receptor point mutation affecting interaction with phospholipase C-gamma 1 abolishes NGF-promoted peripherin induction but not neurite outgrowth.

We analyzed the function of Trk nerve growth factor (NGF) receptors containing a point mutation (Tyr-->Phe) in a major autophosphorylation site (Tyr-785). Tyr-785 is required for phospholipase C-gamma 1 to interact with Trk and to become tyrosine-phosphorylated in response to NGF. The altered receptors were transfected into a mutant subline of PC12 rat pheochromocytoma cells (designated PC12nnr5) that, unlike wild-type PC12 cells, lack expression of endogenous Trk and responsiveness to NGF. PC12nnr5 cells permanently transfected with Trk Y785F exhibit NGF-dependent autophosphorylation and normal NGF binding and internalization. Moreover, Trk Y785F mediates NGF-stimulated neurite outgrowth as well as a variety of additional responses including induction of immediate-early and late genes. However, in contrast to cells expressing wild-type Trk, cells expressing Trk Y785F lack NGF-promoted elevation of peripherin intermediate filament mRNA and protein. These observations indicate that phospholipase C-gamma 1 activation or other signaling pathways dependent on Tyr-785 autophosphorylation are selectively required for regulation of peripherin expression by NGF, but not for many other functional NGF responses. This supports the presence of multiple and separable signaling pathways in the NGF mechanism of action.

Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses.

In response to NGF, the Trk receptor tyrosine kinase forms a complex with SHC, a protein that couples receptor tyrosine kinases to p21ras. Complex formation between Trk and SHC, SHC tyrosine phosphorylation, and association of SHC with Grb2 were mediated by autophosphorylation at Y490 in Trk [sequence: see text]. To determine the role of SHC and other Trk substrates in NGF signaling, Trk receptors with mutations in Y490 and Y785 (the PLC-gamma 1 association site) were introduced into PC12nnr5 cells. NGF treatment of PC12nnr5 cells expressing Trk with mutations in either substrate-binding site resulted in normal neurite outgrowth and Erk1 activity and tyrosine phosphorylation. However, PC12nnr5 cells expressing Trk with mutations at both sites failed to stably extend neurites and efficiently induce Erk1 activity and tyrosine phosphorylation in response to NGF. We postulate that Trk receptors can activate Erk1 by either SHC- or PLC-gamma 1-dependent signaling pathways. These results suggest a model whereby Trk receptors utilize at least partially redundant signal transduction pathways to mediate NGF responses.

A purine analog-sensitive protein kinase activity associates with Trk nerve growth factor receptors.

Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (+/- NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and approximately 50-80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk-associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity with Trk NGF receptors.

Transfection with trk restores "slow" NGF binding, efficient NGF uptake, and multiple NGF responses to NGF-nonresponsive PC12 cell mutants.

NGF binds to and activates the protein tyrosine kinase gp 140prototrk. Expression of this receptor is required for at least some responses to NGF. Three outstanding issues are addressed in the present work. First, we determined whether expression of gp 140prototrk is required for all neuronal NGF responses. Second, we examined the role of gp 140prototrk in NGF binding and internalization. Third, we addressed the utility of NGF-nonresponsive PC12nnr5 cells for study of the NGF mechanism. In contrast to wild-type PC12 cells, PC12nnr5 cells do not express endogenous gp 140prototrk. We therefore asked whether they possess other defects that compromise NGF signaling pathways. To answer these questions, we transfected PC12nnr5 cells with a cDNA encoding full-length human gp 140prototrk and isolated cell lines permanently expressing the receptor. Introduction of trk rescued all of the many and varied NGF responses assessed, including enhanced protein tyrosine phosphorylation, induction of immediate-early and neural-specific genes, neurite outgrowth and regeneration, maintenance of survival in serum-free medium, and stimulation of AChE activity. In contrast to PC12nnr5 cells, the trk-transfected lines also bind and internalize NGF with wild-type PC12 cell characteristics. These findings indicate that gp 140prototrk is required for many, if not all, responses of neuronal cells to NGF and is necessary for proper NGF binding and internalization. Additionally, as no signaling defect other than the absence of trk expression was revealed in PC12nnr5 cells, this work supports the utility of this line for genetic dissection of the NGF mechanism of action.

NGF and other growth factors induce an association between ERK1 and the NGF receptor, gp140prototrk.

As detected by coimmunoprecipitation from PC12 cells, NGF induces rapid association between ERK1 (a growth factor-activated serine/threonine protein kinase) and gp140prototrk NGF receptors. In contrast, no such association is found with the closely related ERK2. Anti-trk immunocomplexes generated from NGF-treated cells also contain protein kinase activity that shares many properties with soluble ERK1. The association of both ERK1 protein and ERK-like kinase activity with gp140prototrk is maximal by 5 min of NGF treatment, persists for approximately 1 hr, and subsequently declines by 18 hr. Treatment with either basic fibroblast growth factor, epidermal growth factor, or orthovanadate also leads to association of ERK1 with gp140prototrk without tyrosine phosphorylation of the latter. The interaction between ERK1 and gp140prototrk may prove relevant to the NGF mechanism.

The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines.

The trk tyrosine kinase proto-oncogene product gp140prototrk binds nerve growth factor (NGF) and is rapidly and selectively activated by this neurotrophic factor. To determine whether gp140prototrk is involved in transducing a functional NGF signal, PC12 cell mutants (PC12nnr) deficient in high affinity NGF binding and unresponsive to NGF were used. Northern analysis revealed that these mutant cells have greatly reduced levels of trk expression. PC12nnr cultures were transiently transfected with expression vectors encoding the full-length rat trk cDNA and assessed for responsiveness to NGF. Expression of exogenous trk rescued the capacity for NGF-promoted neurite outgrowth, cellular hypertrophy, and serum-free survival by these cells. These results indicate that gp140prototrk is necessary for functional NGF signal transduction.