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L J Helman

Publications and source records attributed to L J Helman.

At least 19 recordsLinked to original sources
PubMed2006

Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism.

Rapamycin and several analogs, such as CCI-779 and RAD001, are currently undergoing clinical evaluation as anticancer agents. In this study, we show that inhibition of mammalian target of rapamycin (mTOR) signaling by rapamycin leads to an increase of Akt phosphorylation in Rh30 and RD human rhabdomyosarcoma cell lines and xenografts, and insulin-like growth factor (IGF)-II-treated C2C12 mouse myoblasts and IGF-II-overexpressing Chinese hamster ovary cells. RNA interference-mediated knockdown of S6K1 also results in an increase of Akt phosphorylation. These data suggest that mTOR/S6K1 inhibition either by rapamycin or small interfering RNA (siRNA) triggers a negative feedback loop, resulting in the activation of Akt signaling. We next sought to investigate the mechanism of this negative feedback regulation from mTOR to Akt. Suppression of insulin receptor substrate (IRS)-1 and tuberous sclerosis complex-1 by siRNAs failed to abrogate rapamycin-induced upregulation of Akt phosphorylation in both Rh30 and RD cells. However, pretreatment with h7C10 antibody directed against insulin-like growth factor-1 receptor (IGF-1R) led to a blockade of rapamycin-induced Akt activation. Combined mTOR and IGF-1R inhibition with rapamycin and h7C10 antibody, respectively, resulted in additive inhibition of cell growth and survival. These data suggest that rapamycin mediates Akt activation through an IGF-1R-dependent mechanism. Thus, combining an mTOR inhibitor and an IGF-1R antibody/inhibitor may be an appropriate strategy to enhance mTOR-targeted anticancer therapy.

Animals↗
PubMed2001

Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

Amino Acid Sequence↗
PubMed2001

Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.

PURPOSE: A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing's sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing's sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing's sarcoma in such specimens. PATIENTS AND METHODS: Binding of monoclonal antibody (MoAb) 8H9 to Ewing's sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR--based amplification of t(11;22), levels of Ewing's contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays. RESULTS: MoAb 8H9 shows binding to Ewing's sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing's sarcoma cells with a sensitivity of one cell in 10(6) normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing's sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10(6). Levels of contamination in clinical samples ranged from 1:10(5) to 10(6). Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR. CONCLUSION: These results demonstrate a new approach for purging contaminated cellular products of Ewing's sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease.

Animals↗
PubMed2000

XAGE-1, a new gene that is frequently expressed in Ewing's sarcoma.

Our previous expressed sequence tag database analysis indicates that XAGE-1 is frequently found in Ewing's sarcoma and alveolar rhabdomyosarcoma (U. Brinkmann et al., Cancer Res., 59: 1445-1448, 1999). Using Northern blots and RNA dot blots, we have now found that XAGE-1 is highly expressed in normal testis, in seven of eight Ewing's cell lines, in four of nine Ewing's sarcoma patient samples, and in one of one alveolar rhabdomyosarcoma patient sample. The gene is located on the X chromosome. The full-length cDNA contains 611 bp and predicts a protein of Mr 16,300 with a potential transmembrane domain at the NH2 terminus. XAGE-1 shares homology with GAGE/PAGE proteins in the COOH-terminal end. These findings could be valuable for cancer diagnosis and cancer immunotherapy.

Amino Acid Sequence↗
PubMed2000

Inguinal hernia in patients with Ewing sarcoma: a clue to etiology.

BACKGROUND: Various congenital anomalies have been associated with childhood cancer, but as yet no anomaly has been consistently found with Ewing sarcoma (ES). Recently a large case-control study of ES patients reported a greater number of hernias in both cases and their sibling controls than in population controls. Most of these hernias were inguinal. Because these anomalies were also reported previously in two case series, we looked for inguinal hernias in a different population of ES patients. PROCEDURE: We abstracted medical records for 306 pathologically confirmed ES/primitive neuroectodermal tumor (PNET) patients seen at NIH between 1960 and 1992. Epidemiological data on demographics and medical conditions were analyzed. The frequency of anomalies was compared to expected rates to calculate relative risk and confidence intervals. RESULTS: Anomalies were present in 67 (22%) cases. A particular anomaly, inguinal hernia, was reported for 13 (5%) NIH cases. Compared to population estimates for white children, the relative risk of inguinal hernia among white NIH cases was 13.3 (95% CI 3.60-34.1) for females and 6.67 (95% CI 2.67-13.7) for males. CONCLUSIONS: The findings of inguinal hernias in some patients with ES suggest that a disruption in normal embryological development occurred. This may provide an important clue to the etiology of ES. We hypothesize that these hernias may relate to an in utero exposure or indicate an underlying genetic disorder. Future studies should carefully evaluate ES families for genetic disease and explore environmental factors. Med. Pediatr. Oncol. 34:195-199, 2000. Published 2000 Wiley-Liss, Inc.

Adolescent↗
PubMed2000

Targeting pediatric malignancies for T cell-mediated immune responses.

Successful immune targeting of malignancies hinges upon the ability to activate specific T-cell populations to recognize and attack tumor but spare normal vital tissues. Investigators in the field of tumor immunology are currently utilizing at least three distinct approaches toward this goal. In the first approach, molecular targets of cytolytic T cells which spontaneously develop in tumor-bearing patients have been identified and are subsequently used as immunogens in immunotherapy trials. Whereas this approach originally focused upon the identification of tumor antigens in the immune-responsive tumors malignant melanoma and renal cell carcinoma, it surprisingly led to the identification of a variety of molecules that are now known to be expressed in other common pediatric and adult tumors. In the second approach, tumor-specific molecules (eg, mutant p53 and chromosomal translocations) that have been identified in individual tumors during the study of neoplastic transformation are used as immunogens. Because chromosomal translocations are common in pediatric tumors, such targets may be of particular interest in pediatric oncology. In the third approach, immunization with whole tumor cell components is undertaken with the assumption that the most immunogenic molecules within the tumor will dominate the immune response induced. The benefits and limitations for each approach, particularly as it pertains to the development of immunotherapy for pediatric tumors, are discussed in this article.

Antigens, Neoplasm↗
PubMed2000

Targeting tumor specific translocations in sarcomas in pediatric patients for immunotherapy.

In an effort to develop more effective therapies for various sarcomas in pediatric patients, the authors have focused on using recurrent tumor-specific translocations as potential novel tumor antigens. In general, these translocations generate fusion transcription factors. Because cytotoxic T cell lymphocyte receptors recognize peptide fragments bound to major histocompatibility complex Class 1 molecules, it is possible that unique peptides spanning the translocation breakpoint region may be processed, bound to major histocompatibility complex Class I molecules and displayed on the tumor cell surface where they could be susceptible to cytotoxic T cell lymphocyte killing. The authors have investigated the PAX-3-FKHR fusion product seen in alveolar rhabdomyosarcoma, and the EWS-FLI-1 fusion product seen in Ewing's sarcoma. Peptides spanning these fusion regions contain potential major histocompatibility complex Class 1 and Class II binding motifs suggesting they may serve as novel T cell antigens. Preliminary mouse experiments suggest that cytotoxic T cell lymphocytes specific for the PAX-3-FKHR fusion peptide can be generated and can recognize and kill tumor cells bearing the PAX-3-FKHR fusion protein. Clinical trials are ongoing to determine whether this approach will be useful.

Animals↗
PubMed1999

Rhabdomyosarcoma--working out the pathways.

Rhabdomyosarcomas constitute a collection of childhood malignancies thought to arise as a consequence of regulatory disruption of skeletal muscle progenitor cell growth and differentiation. Our understanding of the pathogenesis of this neoplasm has recently benefited from the study of normal and malignant myogenic cells in vitro, facilitating the identification of diagnostic cytogenetic markers and the elucidation of mechanisms by which myogenesis is regulated. It is now appreciated that the delicate balance between proliferation and differentiation, mutually exclusive yet intimately associated processes, is normally controlled in large part through the action of a multitude of growth factors, whose signals are interpreted by members of the MyoD family of helix - loop - helix proteins, and key regulatory cell cycle factors. The latter have proven to be frequent targets of mutational events that subvert myogenesis and promote the development of rhabdomyosarcoma. Although significant progress has been made in the treatment of rhabdomyosarcoma, patients presenting with metastatic disease or certain high risk features are still faced with a dismal prognosis. Only now are genetically engineered mouse models becoming available that are certain to provide fresh insights into the molecular/genetic pathways by which rhabdomyosarcomas arise and progress, and to suggest novel avenues of therapeutic opportunity.

Animals↗
PubMed1999

Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression.

High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After gamma-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following gamma-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis.

Animals↗
PubMed1998

AP-2 may contribute to IGF-II overexpression in rhabdomyosarcoma.

The human insulin-like growth factor II gene is regulated in a development-dependent manner and is not expressed in most adult tissues. However, high levels of insulin-like growth factor II mRNA are detected in many human tumors including rhabdomyosarcoma, an embryonal tumor of skeletal muscle origin. In this study, we demonstrate that the developmentally regulated transcription factor AP-2 is expressed at higher levels in human fetal skeletal muscle and rhabdomyosarcoma cells compared to human adult skeletal muscle. Endogenous insulin-like growth factor II mRNA derived from the P3 as well as transfected P3 promoter activity were modestly and consistently increased to the same extent following treatment of the rhabdomyosarcoma cell line RD with forskolin, a compound implicated in AP-2 transactivation. This effect of AP-2 on increased transcriptional activity was confirmed by nuclear run-on assays. Expression of AP-2B, a dominant-negative inhibitor of AP-2, suppressed the P3 promoter activity in AP-2 expressing RD cells. Furthermore, five AP-2 protected regions corresponding to six AP-2 specific binding sites were detected in the insulin-like growth factor II P3 promoter. These data together suggest that AP-2 may contribute to the high expression of IGF-II in rhabdomyosarcoma cells.

Binding Sites↗
PubMed1998

Biallelic expression of all four IGF-II promoters and its association with increased methylation of H19 gene in human brain.

The human IGF-II gene is maternally imprinted in all tissues except adult liver and the choroid plexus/leptomeninges of the central nervous system where IGF-II is biallelically expressed. In human liver, it has recently been reported that this biallelic expression only involves the promoter P1 while the promoters P2-P4 direct IGF-II transcription monoallelically. To explore whether or not biallelic expression of the IGF-II promoters in human CNS displays the same pattern as in liver, we examined the allelic expression status of the four IGF-II promoters in human brain. We found that all four IGF-II promoters in human fetal and adult brain were expressed from both parental alleles. Furthermore, the levels of methylation of the 3' region of H19 gene in fetal brain were higher than those in other tissues with monoallelic expression of IGF-II. Since similar findings have been reported in Wilms' tumor, these data suggest a similar mechanism may be responsible for loss of imprinting of IGF-II in normal brain and Wilms' tumor.

Adult↗
PubMed1998

Expression of a kinase-deficient IGF-I-R suppresses tumorigenicity of rhabdomyosarcoma cells constitutively expressing a wild type IGF-I-R.

Previous results have shown that the insulin-like growth factor type I receptor (IGF-I-R) plays a critical role in the control of rhabdomyosarcoma (RMS) growth. The purpose of this study was to investigate whether a mutated IGF-I-R, when expressed in RMS cells, may interfere with the function of the endogenous wild-type IGF-I-R. We also examined whether the expression of a mutated IGF-I-R may induce phenotypic changes in RMS cells. We used here the mutated IGF-I-R with a lysine to arginine residue 1003 substitution, called IGF-I-KR, which carries a mutation in the ATP-binding domain of the intracellular beta subunit, while the extracellular, ligand binding alpha subunit remains unchanged. We observed that the expression of this mutated IGF-I-KR markedly decreased the response of RMS cells to stimulation with IGF-I. While stimulation with IGF-I increases the autophosphorylation of IGF-I-R in the parent cells, stimulation with IGF-I failed to produce a comparable increase in autophosphorylation in the cells expressing the mutated IGF-I-KR. We also observed a decreased plating efficiency of cells expressing the mutated IGF-I-KR. Consistently, a decrease of RMS growth in vivo was observed in an animal model. Our data suggest that the IGF/IGF-I-R signaling pathway may be inhibited by expressing a mutated IGF-I-KR and that such a mutant gene could be utilized in developing novel therapeutic strategies to suppress RMS growth. 1998.

Cell Division↗
PubMed1998

p53 regulates human insulin-like growth factor II gene expression through active P4 promoter in rhabdomyosarcoma cells.

The developmentally regulated human insulin-like growth factor II (IGFII) gene is expressed at high levels in many types of tumors and promotes the proliferation of tumor cells with a high incidence of p53 gene defects. We have previously shown that p53 inhibits IGFII P3 promoter activity and decreases endogenous IGFII gene expression derived from the P3 promoter in rhabdomyosarcomas by interfering with TBP binding to the TATA element of the IGFII P3 promoter. In this report, we demonstrate that wild-type p53 expression in rhabdomyosarcoma cell lines containing mutant p53 leads to a decrease in the activity of another active IGFII promoter, P4, and a 5-fold reduction of IGFII mRNA derived from the P4 promoter. This inhibition of P4 activity is associated with direct binding of p53 to the P4 proximal promoter element despite the lack of a p53 consensus binding site. Our results suggest that p53 inhibits IGFII P4 promoter activity by a mechanism different than its effect on the P3 promoter. These data also supply further evidence of cross-talk between the IGF and p53 signaling pathways.

DNA, Neoplasm↗
PubMed1997

The insulin-like growth factor-I receptor is required for EWS/FLI-1 transformation of fibroblasts.

Ewing's family of tumors is characterized by a well described reciprocal translocation, t(11;22)(q24;q12), which produces a fusion protein (EWS/FLI-1) that transforms mouse fibroblasts. The EWS/FLI-1 fusion protein has been shown to act as a potent chimeric transcription factor. Overexpression of insulin-like growth factor-I receptor (IGF-IR) has been implicated in many tumor models as playing a role in cell growth and tumorigenesis. In addition, blockade of the IGF-IR inhibits the growth of Ewing's family of tumors cells. Therefore, we first studied whether the presence of the IGF-IR is required for transformation by the EWS/FLI-1 fusion protein. To perform this study, we used two previously described fibroblast cell lines, R- and W, derived from an IGF-IR knockout mouse and a wild-type littermate, respectively. Neither W nor R- cells without the fusion protein formed soft agar colonies. However, W clones expressing the fusion message (WF cells) formed soft agar colonies, whereas R- clones expressing the fusion message (R-F cells) did not form soft agar colonies. Because the IGF-IR is required for EWS/FLI-1 transformation, we chose to investigate whether altered signaling occurs from the IGF-IR when the EWS/FLI-1 fusion is present. WF cells demonstrated a greater degree of ligand-stimulated insulin receptor substrate-1 phosphorylation when compared with W cells, suggesting that expression of the EWS/FLI-1 fusion protein alters the IGF-IR signaling pathway.

Animals↗
PubMed1997

Cytogenetics and experimental models.

The use of cytogenetics has led to significant improvement in the diagnoses and classification of sarcomas. Many of the major sarcomas have been to have characteristic tumor-specific chromosomal translocations that are currently used in the diagnosis of these tumors. In the past year, a subset of Ewing's family of tumors and myxoid liposarcomas, which lack one of the characteristic translocations, were found to carry related translocations. New technologies such as a spectral karyotyping will likely increase out ability to identify additional tumor-specific translocations. The emergence of genetic alterations as prognostic factors, as illustrated by Ewing's family of tumors, osteosarcoma, and p53 expression in soft tissue sarcomas in general, is discussed. The review concludes with laboratory applications derived from either tumor cytogenetic or gene function abnormalities that are related to tumor-specific translocations. It is anticipated that advances in diagnosis, prognosis, and modeling will translate into future therapeutic advances.

Humans↗
PubMed1996

Suppression of rhabdomyosarcoma growth by fumagillin analog TNP-470.

The purpose of our study was to investigate a novel therapeutic approach for rhabdomyosarcoma (RMS) in an animal model. The pursuit of new therapeutic modalities for RMS is critically important since this type of tumor is the most common soft tissue sarcoma in children and because patients with metastatic disease may not be cured with current therapeutic modalities. We studied whether RMS growth may be suppressed by TNP-470, an analog of fumagillin, which was found to inhibit neoangiogenesis. Our data had shown that animals treated with TNP-470 (60 mg/kg), over a specific period of time, had approximately 50% smaller tumors than controls. Consistent with previous observations, treatment with TNP-470 decreases the level of the cyclin D1. Tumors dissected from TNP-470-treated animals had also considerable necrotic areas. In addition, TNP-470 had a direct cytotoxic effect on RMS cells in vitro. Our study has shown, therefore, that RMS in an animal model and in vitro responds to treatment with TNP-470, which suggests that the inhibitors of angiogenesis may be useful in a novel therapeutic design for RMS.

Animals↗