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S L Cohn

Publications and source records attributed to S L Cohn.

At least 55 records · Page 3Linked to original sources
PubMed1997

Modulation of N-myc expression alters the invasiveness of neuroblastoma.

N-myc oncogene expression plays a pivotal role in the biology of neuroblastoma, a common childhood tumor. High N-myc expression is associated with advanced disease stage, and in animal models, increased expression results in increased metastatic potential. In normal embryologic development, N-myc expression is associated with neuroblast migration out from the neural crest. To further define the relationship between N-myc and metastasis, an in vitro assay was adapted to measure tumor cell attachment, motility, and proteolytic ability in neuroblastoma cell lines. These parameters were examined in a non-amplified, uniformly N-myc overexpressing cell line and its anti-sense N-myc expressing clones. These lines have been characterized previously, and have a decrease in N-myc expression, growth rate, and tumorigenicity relative to the parent line and vector-only control transfectant. Decrease in N-myc expression resulted in a non-proportional increase of tumor cell attachment, and a proportional decrease in both tumor cell motility and proteolytic ability. In further experiments, assay of a N-myc-amplified overexpressing cell line with an intrinsic heterogeneous pattern of expression demonstrated that motile cells expressed higher amounts of N-myc relative to the general population. Together these relationships indicate that N-myc plays a causative role in the invasive phenotype, and suggest that metastasis may, in part, result from the disruption of a developmentally important normal process.

Cell Adhesion↗
PubMed1997

Treatment of poor-risk neuroblastoma patients with high-dose chemotherapy and autologous peripheral stem cell rescue.

A single institutional pilot study was conducted in which 12 poor-risk neuroblastoma (NB) patients were uniformly treated with multi-agent induction chemotherapy followed by myeloablative consolidation chemotherapy and unpurged peripheral blood stem cell (PBSC) rescue. In addition to using standard criteria for evaluating response to induction chemotherapy, tumor cell contamination of the peripheral blood and/or bone marrow was analyzed in seven patients by immunocytology using a panel of five anti-NB monoclonal antibodies. Seven patients had morphologic evidence of bone marrow disease at the time of diagnosis, and two additional patients had tumor cells detected in bone marrow samples by immunocytology prior to the second cycle of chemotherapy. After three cycles of chemotherapy, two of the 12 patients continued to have evidence of bone marrow disease. Samples from 29 PBSC harvests collected from nine patients were also analyzed for the presence of contaminating tumor cells by immunocytology. In each case, the stem cells were found to be free of tumor. Eleven of the 12 patients underwent myeloablative therapy and PBSC rescue; five patients remain alive without disease progression, 28+ to 53+ months from diagnosis, and six patients have developed recurrent disease. We conclude that PBSCs can be successfully harvested from children with NB, and used for hematopoietic reconstitution following myeloablative chemotherapy. However, more effective therapy for poor-risk NB patients is still urgently needed.

Adult↗
PubMed1996

Binding of a 40-kDa protein to the N-myc 3'-untranslated region correlates with enhanced N-myc expression in human neuroblastoma.

Subclones of neuronal (N) and non-neuronal (S) cells established from neuroblastoma tumors cultured in vitro differ in their growth characteristics and N-myc expression. N (W-N) cells derived from the NBL-W cell line express 5-fold higher levels of N-myc mRNA and 10-12-fold higher levels of protein than S cells (W-S), despite having the same N-myc copy number. This study demonstrates that the steady-state levels of N-myc are largely determined by differences in N-myc mRNA stability. The half-life of N-myc mRNA in the W-N cells is approximately 35 min compared with approximately 6 min in the W-S cells. Turnover of labile mRNAs is thought to be mediated in part by the interactions of trans-acting factors with elements within the 3'-untranslated region. RNA UV cross-linking assays using W-N cell lysate demonstrate abundant quantities of a protein complex that is 40 kDa in size (p40) that binds to the N-myc 3'-untranslated region. p40 is barely detectable in W-S cells. We have mapped two distinct regions within the 3'-UTR that specifically bind p40 (base pairs 5694-5715 and 6465-6482). Analysis of nine additional neuroblastoma cell lines shows that p40 activity correlates with enhanced expression of N-myc. p40 activity is also detected in 5 of 19 primary neuroblastomas, and activity is associated with clinically aggressive disease. In the accompanying study, we identify p40 as a member of the embryonic lethal abnormal vision (ELAV)-like family of RNA-binding proteins. Our studies suggest that ELAV-like proteins may play a role in the regulation of N-myc mRNA turnover and thereby modulate the steady-state levels of N-myc expression and tumor cell phenotype.

Binding Sites↗
PubMed1996

Differential activity of ELAV-like RNA-binding proteins in human neuroblastoma.

Many short-lived mRNAs contain AU-rich instability elements within their 3'-untranslated region. Cellular factors that bind to these elements are thought to play a role in the regulation of mRNA degradation. In the accompanying paper (Chagnovich, D., and Cohn, S. L. (1996) J. Biol. Chem. 271, 33580-33586) we characterized the binding activity of a 40-kDa protein (p40) that interacts with high specificity with at least two AU-rich elements located within the 3'-untranslated region of N-myc. p40 activity correlates with N-myc mRNA stability in subclones of the NBL-W neuroblastoma cells line (W-N and W-S). In an effort to determine the identity of p40 we performed immunoblotting studies, immunoprecipitation experiments, and RNA gel mobility shift assays using antibodies that are directed against known RNA-binding proteins. In this paper we demonstrate that in W-N and W-S cells, p40 activity parallels the expression of embryonic letal abnormal vision (ELAV)-like proteins, and that antibodies directed against this family of RNA-binding proteins recognize p40. We also show that purified ELAV-like proteins (HuD and Hel-N1) bind with high specificity to the same N-myc 3'-untranslated region sequences as p40. Our data indicate that p40 is a member of the ELAV-like family, and suggest that this family of RNA-binding proteins may regulate N-myc mRNA turnover.

ELAV Proteins↗
PubMed1996

Physical mapping of the DDX1 gene to 340 kb 5' of MYCN.

One of the most important prognostic factors in neuroblastoma is amplification of the MYCN gene, which is strongly associated with advanced stages of disease and a poor prognosis. Although the MYCN amplicon sometimes spans more than 1 Mb, no other consistently expressed sequences from the MYCN amplicon have been reported. However, DDX1, a gene encoding a DEAD box protein, was recently mapped to chromosome 2p24 and is frequently co-amplified with MYCN. Therefore, we performed genomic mapping with YACs to determine the physical relationship between DDX1 and MYCN, and whether DDX1 was contained within the core region of amplification. Based on YAC restriction mapping and content analysis, DDX1 maps 340 kb 5' of MYCN, outside the core domain of consistent amplification. Interestingly, we also determined by sequence analysis and detailed restriction mapping that G21, previously isolated as a 'neuroblastoma-specific' cDNA clone from an MYCN amplicon, is a partial cDNA of DDX1. Our data confirm that DDX1 is amplified in some but not all MYCN-amplified tumors, and that it is rearranged in other cases. This suggests that the co-amplification of DDX1 is due to its proximity to MYCN.

Chromosome Mapping↗
PubMed1996

Analysis of possible WT1 RNA processing in primary Wilms tumors.

WT1 RNA processing abnormalities have been suggested to play a role in the development of Wilms tumor by reports of editing at codon 280 in the rat WT1 transcript (codon 281 in humans) and aberrant splicing of exon 2 in WT1 transcripts from Wilms tumor xenograft cell lines. Both events result in a functionally changed WT1 protein and are potential mechanisms of altering normal protein function in the absence of WT1 DNA mutations. To determine whether either of these RNA processing events occurs in primary Wilms tumors, we analysed WT1 mRNA from 15 primary tumors. There was no evidence of WT1 RNA editing at codon 281, and only one primary tumor displayed aberrant splicing of exon 2. Sequence and Southern analysis of DNA from this tumor did not reveal any alteration in or around exon 2. These results suggest that neither RNA editing at codon 281 nor aberrant exon 2 splicing is a frequent mechanism of WT1 alteration during tumorigenesis.

Alternative Splicing↗
PubMed1996

Expression of the gene for multidrug-resistance-associated protein and outcome in patients with neuroblastoma.

BACKGROUND: Overexpression of the gene for the multidrug-resistance-associated protein (MRP) has been linked with resistance to chemotherapeutic agents (multidrug resistance) in vitro. The expression of MRP by neuroblastoma cells correlates with N-myc oncogene amplification, a well-established prognostic indicator in patients with neuroblastoma. METHODS: To relate MRP gene expression to established prognostic markers and the clinical outcome of neuroblastoma, we analyzed MRP expression in specimens of primary tumors from 60 patients with neuroblastoma. RESULTS: Levels of MRP gene expression were significantly higher in tumors with N-myc amplification than in tumors without such amplification (P < 0.001). High levels of MRP expression were strongly associated with reductions in both survival and event-free survival (P < 0.001) in the overall study population and in subgroups of patients without N-myc amplification and patients with localized disease. For the overall study population, the five-year cumulative survival rates in the groups with high and low levels of MRP expression were 57 percent (95 percent confidence interval, 37 to 78 percent) and 94 percent (95 percent confidence interval, 86 to 100 percent), respectively. In contrast, expression of the MDR1 multi-drug-resistance gene was not predictive of survival or event-free survival. After adjustment by multivariate analysis for the effects of N-myc amplification and other prognostic indicators, high levels of MRP expression retained significant prognostic value for poor survival (relative hazard, 14.9; P = 0.01) and poor event-free survival (relative hazard, 9.7; P = 0.004), whereas N-myc amplification had no prognostic value. CONCLUSIONS: High levels of MRP gene expression in patients with neuroblastoma correlate strongly with poor outcome. The findings suggest that expression of this multidrug-resistance gene accounts for the association between N-myc amplification and reduced survival.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Up-regulation of HOXC6, HOXD1, and HOXD8 homeobox gene expression in human neuroblastoma cells following chemical induction of differentiation.

An early event in the pathogenesis of neuroblastoma (NB), a tumor derived from embryonal neural crest tissue, appears to be the arrested differentiation of neuroblasts. However, NB cells can be induced to differentiate in vitro with numerous chemicals including retinoic acid (RA) and dibutyryl cyclic AMP (db-cAMP). One family of transcription factors, encoded by the homeobox (HOX) genes, plays a crucial role in Drosophila, Xenopus, and mammalian embryonic differentiation and development. We have previously identified six HOX genes (HOXC6, HOXC8, HOXD1, HOXD4, HOXD8, and HOXD9), by a sensitive PCR-based approach, in a cDNA library prepared from the human LA-N-5 NB cell line induced to differentiate with RA. In this report, we studied the regulation of these six HOX genes in a series of NB cell lines chemically induced to differentiate. Untreated NB cells express low or undetectable levels of HOX mRNA, and HOXC8 remains undetectable in the induced cells. However, a significant induction of HOXC6, HOXD1, and HOXD8 expression is seen in the RA-treated NB cell lines, albeit with different patterns and degree of up-regulation. db-cAMP treatment also induced HOXC6 and HOXD8 expression in two of the three NB cell lines analyzed. Low levels of HOXD4 and HOXD9 induction were observed in two and one RA-treated NB cell line, respectively. Up-regulation of HOXC6, HOXD1, and HOXD8 expression in human NB cells, chemically induced to differentiate, appears to be associated with maturation toward a differentiated neuronal phenotype.

Cell Differentiation↗
PubMed1996

Tumor angiogenesis correlates with metastatic disease, N-myc amplification, and poor outcome in human neuroblastoma.

PURPOSE: To determine if the clinical outcome of children with neuroblastoma (NB) is correlated with the degree of tumor neovascularization and to assess the relationship of stage, N-myc copy number, and histology to angiogenesis. MATERIALS AND METHODS: The vascularity of primary untreated NB from 50 patients diagnosed at a single institution between 1984 and 1994 was evaluated. An image processor was used to analyze the tumor tissue area for each histologic slide of tumor, and a vascular index (VI) was calculated, where VI = total number of vessels/mm2 of tissue area. Tumors were classified histologically according to the criteria of Shimada et al (J Natl Cancer Inst 73:405-416, 1984), and N-myc copy number was determined by Southern blot analysis. RESULTS: We found that higher VI (> 4.0) in NB strongly correlated with widely disseminated disease (P = .006) and poor survival (P < .0001). VI more than 4.0 was also statistically associated with N-myc amplification (P = .02) and unfavorable histology (P = .02). Univariate analysis demonstrated that disease stage, tumor histology, and N-myc copy number were also predictive of outcome. Cox regression analysis showed that VI provided independent prognostic information. CONCLUSION: Our studies indicate that angiogenesis may play an important role in determining the biologic behavior of NB. Antiangiogenic therapy may prove to be effective in the treatment of children with highly vascular, widely disseminated NB.

Adult↗
PubMed1996

Loss of DCC expression in neuroblastoma is associated with disease dissemination.

DCC, a candidate tumor suppressor gene from chromosome 18q21, is most highly expressed in the developing nervous system. In vitro studies suggest a role for DCC in neuronal differentiation, and 18q allelic loss occurs in a subset of neuroblastomas. To address the hypothesis that loss of DCC function may contribute to tumorigenesis in cells of neural origin, we utilized a combination of RNase protection, immunoblotting, and immunohistochemical approaches to characterize DCC expression in 62 primary neuroblastomas and 16 neuroblastoma cell lines. The DCC protein was undetectable in 38% of the primary tumors and 56% of the cell lines. Of note, primary tumors lacking DCC expression were more likely to have been obtained from patients with disseminated or stage D disease (P = 0.01). In addition, loss of DCC expression was observed in three of six primary tumors from stage DS patients. No consistent relationship between the loss of DCC expression and N-myc amplification was observed in our studies. Our findings suggest that loss of DCC expression may contribute to the dissemination of neuroblastoma cells, perhaps through alterations in growth and differentiation pathways distinct from those regulated by N-myc.

Cell Adhesion Molecules↗
PubMed1995

Lack of correlation of N-myc gene amplification with prognosis in localized neuroblastoma: a Pediatric Oncology Group study.

Multiple copies of N-myc proto-oncogene are only rarely detected in localized neuroblastomas (NBs), and the prognostic relevance of amplification in this subset of patients is not clear. We analyzed a series of 850 children with NB admitted to a Pediatric Oncology Group NB Biology Study and identified six patients with localized NBs harboring N-myc gene amplification. Three patients whose tumors showed favorable histology by Shimada classification and low-risk histological features according to the Joshi classification have remained disease-free, whereas two of three patients with unfavorable histology tumors have developed recurrent disease. Although earlier studies have indicated that N-myc amplification is associated with diploid DNA content, flow cytometric analysis revealed that only two of the localized tumors contained stem lines with diploid DNA content. Loss of chromosome 1p was not detected by fluorescence in situ hybridization in the two tumors examined. N-myc protein was detected by immunohistochemical studies in four of the five NBs analyzed. However, N-myc protein was not visualized in one of the tumors with stroma-rich histology, and Western blot analysis revealed only low levels of N-myc protein expression in another NB with favorable histology. These studies indicate that the presence of N-myc amplification in localized NBs does not necessarily portend an adverse outcome. Furthermore, the biological features of this subset of N-myc-amplified NBs appear to differ from those of more advanced N-myc-amplified tumors.

Child↗
PubMed1995

Co-amplification and concomitant high levels of expression of a DEAD box gene with MYCN in human neuroblastoma.

MYCN gene amplification is strongly correlated with poor prognosis in neuroblastoma (NB), the second most common solid pediatric tumor. However, increased MYCN expression seen in tumors that lack MYCN amplification does not correlate with aggressive clinical behavior. Whereas the MYCN gene spans only 7 kb, the MYCN amplicon has been shown to range in size from 350 kb to more than 1 Mb. Given the large size of the amplicon, it is possible that additional genes are co-amplified in NBs whose expression may contribute to the aggressive phenotype associated with MYCN-amplified tumors. We isolated a cDNA clone from a human NB library that is identical to DDXI, a gene recently reported to be preferentially expressed in two retinoblastoma cell lines that also express high levels of MYCN. DDXI belongs to a family of genes that encode DEAD (Asp-Glu-Ala-Asp) box proteins, putative ATP-dependent RNA helicases implicated in a number of cellular processes involving alterations of RNA secondary structure. We examined the frequency of DDXI amplification in 15 NB cell lines, 1 neuroepithelioma cell line, and 122 NB tumors by Southern blot analyses, and we found that 7 of 10 MYCN-amplified cell lines and 27 of 40 (68%) MYCN-amplified tumors also harbored multiple copies of the DDXI gene. Amplification of DDXI was associated with high levels of DDXI mRNA expression in the NB cell lines and tumors as examined by Northern analysis. Neither DDXI gene amplification nor enhanced expression was observed in tumors or cell lines that lacked MYCN amplification. Because RNA helicases play important roles in both post-transcriptional and translational gene regulation, high levels of DDXI expression consequent to genomic amplification may contribute to the malignant phenotype of a subset of NBs.

Amino Acid Sequence↗
PubMed1994

Intraspinal Wilms' tumor metastases.

BACKGROUND: Intraspinal Wilms' tumor metastasis is rare, and is associated with a high mortality rate. METHODS: The authors reviewed the clinical course of two patients with Wilms' tumor in whom extradural metastasis developed. In addition, a review of the literature and of patients entered in the National Wilms' Tumor Studies was performed to determine the clinical presentation, treatment, and outcome of other patients with Wilms' tumor with intraspinal metastases. RESULTS: Both of the patients initially had abdominal pain without neurologic deficits. Despite therapy, paraplegia secondary to cord compression from recurrent epidural metastases developed in one patient, although a third remission has been achieved with further chemotherapy. the second patient remains in disease-free remission 25+ months after surgical resection of the extradural spinal tumor, adjuvant chemotherapy and radiation therapy, and autologous bone marrow transplantation. Review of the literature and of the patients entered in the National Wilms' Tumor Studies revealed an additional 27 patients with Wilms' tumor with this pattern of metastasis. Only four were disease-free at the time of this report. CONCLUSIONS: The authors' experience stresses the importance of early recognition and treatment of this complication of Wilms' tumor and demonstrates that intensive multimodality therapy can result in long-term disease-free remission.

Child↗
PubMed1994

Long-term outcome in children with opsoclonus-myoclonus and ataxia and coincident neuroblastoma.

We reviewed the neurologic and developmental courses in 10 children with opsoclonus-myoclonus ("dancing eyes syndrome") and neuroblastoma. All patients are alive without evidence of neoplastic disease after 8+ to 111+ months of follow-up. All had localized disease and 50% had extraabdominal tumors. Neuroblastomas of nine children had favorable Shimada histologic characteristics, and all tumors had single copies of the N-myc oncogene. After neuroblastoma resection, all patients had persistent opsoclonus-myoclonus or ataxia that responded to therapy with adrenocorticotropic hormone. Nine children had relapses of neurologic symptoms. Three years after resection, six of seven patients with sufficient follow-up were free of symptoms and had discontinued therapy. However, nine children had chronic neurologic deficits, including cognitive and motor delays, language deficits, and behavioral abnormalities. All six patients in educational programs required special assistance. Five children required physical, occupational, or speech therapy. Long-term developmental and cognitive problems should be anticipated in patients with neuroblastoma who have opsoclonus-myoclonus or ataxia or both, and early intervention should be instituted to try to minimize these deficits.

Abdominal Neoplasms↗
PubMed1994

Preferential amplification of the paternal allele in neuroblastomas with N-myc amplification.

There is increasing evidence that imprinting plays an important role in influencing the parental origin of genes involved in cancer-specific rearrangements. In advanced stage neuroblastomas, both allelic loss of the short arm of chromosome 1 (1p) and amplification of the proto-oncogene N-myc are often seen. Therefore, we analyzed 22 human neuroblastomas with N-myc amplification to determine the parental origin of the N-myc allele that is amplified and the 1p allele that is deleted. We used at least three polymorphisms for both the 1p and the N-myc locus to analyze blood and tumor samples from neuroblastoma patients, as well as blood samples from their parents. We determined that the paternal allele of N-myc was amplified in 12 of 15 informative cases (P = 0.02), and the paternal allele on 1p was lost in 6 of 11 informative cases (P > 0.2). These results suggest that parental imprinting does not appear to affect the 1p allele that is deleted, at least in the cases that we have examined. However, imprinting has an important influence on determining the N-myc allele that is amplified in these tumors, suggesting that the paternal allele is either more highly expressed or more likely to undergo amplification.

Alleles↗
PubMed1994

Prenatal detection of neuroblastoma by fetal ultrasonography.

PURPOSE: We report three cases of neuroblastoma diagnosed by prenatal ultrasound examination and examine the biologic features of tumors diagnosed prenatally. PATIENTS AND METHODS: Neuroblastoma is the most common tumor detected in the newborn period. Thus, some of these tumors develop prenatally and should be detectable by maternal ultrasound. Here we report three cases in which a neuroblastoma was suspected on prenatal ultrasonography. In addition, we review selected features of 17 additional cases reported in the literature. RESULTS AND CONCLUSIONS: These data indicate that, although the majority of patients have favorable clinical and biological features and do well, some patients do not, and the DNA index may be the most important predictor of outcome.

Female↗
PubMed1994

The biological effects of antisense N-myc expression in human neuroblastoma.

Although N-myc amplification and overexpression are believed to play an important role in determining the clinical behavior of neuroblastoma (NB), the exact function of N-myc in NB cell growth and differentiation remains unknown. To better understand the function of N-myc, an established human NB cell line was transfected with N-myc antisense (AS) complementary DNA in an effort to down-regulate N-myc gene expression. Five clones expressing AS N-myc RNA have been maintained in culture for over 2 years. Compared to control cells, a 30-69% decrease in the quantity of N-myc protein was demonstrated by Western blot analysis in 4 of the 5 AS clones. All 5 of the AS clones exhibited a 50-75% decrease in colony formation in soft agar assays compared to control cells. In addition, all 5 AS clones expressed a 3.2-kilobase protein kinase C-alpha transcript, whereas this message was not detected by Northern blot analysis in any of the control clones. These results suggest that N-myc may play an important role in NB cell growth and that antisense N-myc expression is associated with an induction of protein kinase C-alpha RNA expression. Further characterization of the AS clones may provide insight into the function of N-myc and may thus lead to a better understanding of the role that N-myc plays in determining the clinical behavior of this childhood neoplasm.

Blotting, Northern↗