Biomedical subjects

B G Feuerstein

Publications and source records attributed to B G Feuerstein.

At least 37 records · Page 2Linked to original sources

PubMed1997

Analyses of brain tumor cell lines confirm a simple model of relationships among fluorescence in situ hybridization, DNA index, and comparative genomic hybridization.

Several techniques are commonly used for genetic analysis of interphase nuclei. Flow cytometry assays the distribution of DNA content in populations of nuclei stained with a DNA-specific fluorochrome. Fluorescence in situ hybridization (FISH) quantifies the number of copies of a specific DNA sequence in single nuclei. Comparative genomic hybridization (CGH) assesses the relative copy number of DNA sequences throughout a test genome by comparing the signal intensities of test and reference DNA samples hybridized to a template of normal metaphase chromosomes. In principle, there are specific relationship among data obtained from these measurements, and combined measurements should provide a more comprehensive view of the sample that is analyzed. We applied these three techniques to nine brain tumor cell lines and find that a model of CGH that includes unsuppressed repeat sequences describes the data well. We estimate that up to 35% of the fluorescence intensity in well-blocked CGH preparations may not represent unique sequences. Taking these factors into account, our results are, in general, mutually consistent, and highlight issues critical for interpreting CGH preparations.

Algorithms↗

PubMed1997

Intraoperative discovery of neuroblastoma in an infant with pulmonary atresia.

There have been 28 previously reported cases of neuroblastoma associated with congenital heart disease. Because many of these have been defects of the conotruncal region, it has been proposed that abnormal neural crest cell migration or maturation may be a factor that links these normally disparate pathologic conditions. Most neuroblastomas in these cases have been detected at autopsy or by radiologic studies conducted in the evaluation of the cardiac anomalies. Recently, we discovered an occult posterior mediastinal neuroblastoma in a patient undergoing a unifocalization procedure for tetralogy of Fallot with pulmonary atresia and major aortopulmonary collaterals. The tumor was resected, and the patient has demonstrated no evidence of residual or metastatic neuroblastoma.

Female↗

PubMed1997

Detection of p16 gene deletions in gliomas: a comparison of fluorescence in situ hybridization (FISH) versus quantitative PCR.

The p16 protein plays a key role in cell cycle control by preventing CDK4 from inactivating the retinoblastoma protein (pRb). The corresponding tumor suppressor gene (p16/MTS1/CDKN2) has recently been implicated in malignant progression of astrocytomas and could potentially serve as an important marker for patient prognosis and for guiding specific therapeutic strategies. We have undertaken a study to evaluate 2 methods of detecting p16 deletion. Thirty diffuse gliomas were analyzed for p16 gene dosage. Dual color fluorescence in situ hybridization (FISH) was performed on cytologic preparations using paired centromeric (CEN) and locus-specific probes for CEN9/p16, CEN8/RB, and CEN12/CDK4. Quantitative PCR was performed using primers for p16, MTAP, and reference genes. Eleven cases were also studied using comparative genomic hybridization (CGH). Abnormalities of the p16-CDK4-RB pathway were identified in 21 (70%) cases by FISH and/or PCR. These included 15 (50%) with p16 deletion, 9 of which were detected by both techniques, 3 by FISH alone, and 3 by PCR alone (concordance rate = 81%). FISH analysis further revealed tetraploidy/aneuploidy in 14 (47%), RB deletion in 11 (37%) and CDK4 amplification in 1 (3.3%). There were 94% and 100% concordance rates between CGH and FISH or PCR, respectively. Quantitative PCR was noninformative in 4 cases. Although FISH and quantitative PCR are both reliable techniques, each has limitations. PCR is likely to miss p16 deletions when there is significant normal cell contamination or clonal heterogeneity, whereas the p16 YAC probe used for FISH analysis may miss small deletions. Replacement of the latter with a cosmid probe may improve the sensitivity of FISH in future experiments.

Brain Neoplasms↗

PubMed1997

Gain of chromosome 17 is the most frequent abnormality detected in neuroblastoma by comparative genomic hybridization.

Neuroblastoma behavior is variable and outcome partially depends on genetic factors. However, tumors that lack high-risk factors such as MYCN amplification or 1p deletion may progress, possibly due to other genetic aberrations. Comparative genomic hybridization summarizes DNA copy number abnormalities in a tumor by mapping them to their positions on normal metaphase chromosomes. We analyzed 29 tumors from nearly equal proportions of children with stage I, II, III, IV, and IV-S disease by comparative genomic hybridization. We found two classes of copy number abnormalities: whole chromosome and partial chromosome. Whole chromosome losses were frequent at 11, 14, and X. The most frequent partial chromosome losses were on 1p and 11q. Gains were most frequent on chromosome 17 (72% of cases). The two patterns of gain for this chromosome were whole 17 gain and 17q gain, with 17q21-qter as a minimal common region of gain. Other common gains were on chromosomes 7, 6, and 18. High level amplifications were detected at 2p23-25 (MYCN region), at 4q33-35, and at 6p11-22. Chromosome 17q gains were associated with 1p and/or 11q deletions and advanced stage. The high frequency of chromosome 17 gain and its association with bad prognostic factors suggest an important role for this chromosome in the development of neuroblastoma.

Child, Preschool↗

PubMed1996

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells.

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.

Becaplermin↗

PubMed1996

Patterns of epidermal growth factor receptor amplification in malignant gliomas.

Amplification of the gene for epidermal growth factor receptor (EGFR) is a common finding in malignant gliomas. We found that 18 of 29 grade 3 and grade 4 gliomas had EGFR amplification when assayed using fluorescence in situ hybridization. The amplification pattern suggests that the amplicon is contained within double minute chromosomes in most cases. EGFR copy number can differ by 20-fold in amplified cells within a single case. Polysomy 7 occurs frequently in both EGFR-amplified and -unamplified cells. More than one-third of the cases had < or = 10 percent of cells with amplified EGFR, and it is likely that these cases would not have been identified by methods that do not examine DNA on a cell by cell basis.

Brain Neoplasms↗

PubMed1995

Spermine uptake is necessary to induce haemoglobin synthesis in murine erythroleukaemia cells.

To determine whether intracellular uptake of spermine is necessary to induce haemoglobin synthesis in murine erythroleukaemia (MEL) DS 19 cells, we used single-step selection for resistance to N1,N12-bis(ethyl)spermine (BESM), a cytotoxic spermine analogue, to isolate clones deficient in polyamine transport. The cells were approximately 500-fold more resistant to BESM than parental cells and were unable to accumulate BESM, putrescine, spermidine or spermine. Addition of spermine to the polyamine-transport-deficient cells failed to induce haemoglobin synthesis. Hexamethylene-1,6-bisacetamide, a well-known differentiating agent, induced haemoglobin synthesis in both parental and resistant cells. Polyamine-transport-deficient cells transfected with DNA purified from the parental cell line were further selected for their ability to grow in the presence of alpha-difluoromethylornithine and putrescine. The transfectants had an active transport system for polyamines, and spermine added to their culture medium accumulated inside the cells and induced haemoglobin production. These findings indicate that intracellular spermine uptake is required to induce haemoglobin production in MEL cells.

Animals↗

PubMed1995

A new model for disruption of the ornithine decarboxylase gene, SPE1, in Saccharomyces cerevisiae exhibits growth arrest and genetic instability at the MAT locus.

Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination.

Blotting, Southern↗

PubMed1995

ERBB-2 (HER2/neu) gene copy number, p185HER-2 overexpression, and intratumor heterogeneity in human breast cancer.

Amplification of the ERBB-2 (HER-2/neu) gene is accompanied by overexpression of its cell surface receptor product, p185HER-2. Heterogeneity has been observed for both the gene copy number and the level of overexpression of its protein product. To better understand their relationship, correlation between the level of cellular expression of p185HER-2 and ERBB-2 gene amplification was studied in four human breast cancer cell lines (BT-474, SK-BR-3, MDA-453, and MCF-7) and in a primary human breast tumor sample. The relative expression of p185HER-2 was measured by immunofluorescence by using flow and/or image cytometry while correlated DNA analysis was performed on the same cells by fluorescence in situ hybridization to determine ERBB-2 gene and chromosome 17 copy numbers. Marked heterogeneity was observed in both protein expression and ERBB-2 copy number. Despite this heterogeneity, and in accordance with previous studies, the average levels of p185HER-2 expression correlated well with average ERBB-2 gene copy numbers in the four lines examined (r = 0.99). When the relationship between copy number and protein expression was studied on a cell-by-cell basis, p185HER-2 expression correlated with both the absolute number of ERBB-2 gene copies/cell (r = 0.59-0.63) and chromosome 17 copy number (r = 0.45-0.61). It is of interest that there was weak or no correlation between p185HER-2 protein expression and the ERBB-2 copy number:chromosome 17 copy number ratio (r = 0.0-0.25). In more than one-half of cells expressing a high level of p185HER-2, the chromosome 17 copy number was high (two or three times the average copy number), whereas < 2% of an unselected population had a high chromosome 17 copy number. Bromodeoxyuridine incorporation indicated that the S-phase-labeling index was homogeneous across various p185HER-2-expressing subpopulations in the SK-BR-3 cell line. Analysis of the primary breast tumor sample showed results similar to the cell lines, supporting the strong possibility of a mechanistic link among p185HER-2 overexpression, ERBB-2 amplification, and high chromosome 17 copy number.

Breast Neoplasms↗

PubMed1995

Spermine induces haemoglobin synthesis in murine erythroleukaemia cells.

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. Haemoglobin production was accompanied by accumulation of cytoplasmic beta-globin mRNA and growth inhibition, but not by cell-cycle block or changes in cell volume. Hexamethylene-bisacetamide (HMBA), a well known differentiating agent, also induces haemoglobin production, but causes a G1 block and decreases cell volume. These findings indicate that HMBA and spermine affect MEL cells differently, even though both induce haemoglobin production.

Acetamides↗

PubMed1995

The structure of polyamine analogues determines haemoglobin production and cytotoxicity in murine erythroleukaemia cells.

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. We have studied the ability of various polyamine analogues to inhibit cell growth and induce haemoglobin production. Polyamine analogues with free terminal amino groups were good inducers of haemoglobin production in MEL cells. Haemoglobin levels correlated with the number of positive charges: pentamines (five positive charges) were stronger inducers than tetramines (four positive charges). Compounds ethylated at their terminal amines were poor inducers of haemoglobin production but good inhibitors of MEL cell growth. These results provide evidence that polyamine analogues support specific biological functions of polyamines in MEL cells and suggest relationships between polyamine structure and function.

Animals↗

PubMed1995

Chromosomal abnormalities in glioblastoma multiforme tumors and glioma cell lines detected by comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a recent molecular cytogenetic method that detects and localizes gains or losses in DNA copy number across the entire tumor genome. We used CGH to examine 9 glioma cell lines and 20 primary and 10 recurrent glioblastoma tumors. More than 25% of the primary tumors had gains on chromosome 7; they also had frequent losses on 9p, 10, 13 and Y. The losses on chromosome 13 included several interstitial deletions, with a common area of loss of 13q21. The recurrent tumors not only had gains on chromosome 7 and losses on 9p, 10, 13 and Y but also frequent losses on 6 and 14. One recurrent tumor had a deletion of 10q22-26. Cell lines showed gains of 5p, 7 and Xp; frequent amplifications at 8q22-24.2, 7q21-32 and 3q26.2-29 and frequent losses on 4, 10, 13, 14 and Y. Because primary and recurrent tumors and cell lines showed abnormalities of DNA copy number on chromosomes 7, 10, 13 and Y, these regions may play a fundamental role in tumor initiation and/or progression. The propensity for losses on chromosomes 6 and 14 to occur in recurrent tumors suggests that these aberrations play a role in tumor recurrence, the development of resistance to therapy or both. Analysis of common areas of loss and gain in these tumors and cell lines provides a basis for future attempts to more finely map these genetic changes.

Chromosome Aberrations↗

PubMed1995

Radiation-induced changes in nucleoid halo diameters of aerobic and hypoxic SF-126 human brain tumor cells.

Nucleoid halo diameters were measured to assay changes in DNA supercoiling in human brain tumor cell line SF-126 after irradiation under aerobic or hypoxic conditions. In unirradiated aerobic cells, a typical propidium iodide titration curve showed that with increasing concentrations of propidium iodide, the halo diameter increased and then decreased with the unwinding and subsequent rewinding of DNA supercoils. In irradiated cells, the rewinding of DNA supercoils was inhibited, resulting in an increased halo diameter, in a radiation dose-dependent manner. To produce equal increases in halo diameter required about a threefold higher radiation dose in hypoxic cells than in aerobic cells. Quantitatively similar differences in the radiation sensitivities of hypoxic and aerobic cells were demonstrated by a colony-forming efficiency assay. These findings suggest that the nucleoid halo assay may be used as a rapid measure of the inherent radiation sensitivity of human tumors.

Aerobiosis↗

PubMed1995

Detection of multiple gains and losses of genetic material in ten glioma cell lines by comparative genomic hybridization.

A protocol for comparative genomic hybridization by use of nucleotides directly labeled with fluorochromes was used to map regions of deletion and amplification in ten glioma cell lines. The protocol greatly reduced experimental artifacts. We detected several genetic aberrations, including whole chromosome loss and gain, partial loss and gain, possible isochromosome, and higher level DNA amplification. The most frequent losses (in order of frequency) occurred on chromosomes 10, 18, 13, 11, 9, 14, 4, 6, 1, and X. The most common gain occurred on chromosome 7. Several sites of previously known and unknown amplifications were observed.

Brain Neoplasms↗

PubMed1995

Two polyamine analogs (BE-4-4-4 and BE-4-4-4-4) directly affect growth, survival, and cell cycle progression in two human brain tumor cell lines.

1,14-Bis-(ethyl)-amino-5,10-diazatetradecane N1,N11-bis(ethyl)norspermine (BE-4-4-4) and 1,19-bis-(ethylamino)-5,10,15 triazanonadecane (BE-4-4-4-4) are two relatively new polyamine analogs synthesized for use as antineoplastic agents. In human brain tumor cell lines U-251 MG and SF-767, both agents inhibited cell growth, were cytotoxic, induced a variable G1/S block, and depleted intracellular polyamines. Since intracellular polyamine depletion did not always correlate with growth inhibition, cell survival, or cell cycle progression, it cannot completely explain the effects of these agents on growth, survival, and cell cycle progression in U-251 MG and SF-767 cells.

Antineoplastic Agents↗

PubMed1995

Molecular cytogenetic quantitation of gains and losses of genetic material from human gliomas.

The unregulated growth that is characteristic of human malignant gliomas is accompanied by, and may result from, losses and/or gains of genetic material. Understanding the mechanisms that underlie how particular genetic aberrations cause dysfunctional growth will help elucidate the pathogenesis of this disease. Two techniques are proving useful in evaluating the clinical relevance of specific genetic aberrations in malignant gliomas. Fluorescence in situ hybridization (FISH) permits direct visualization of gains and losses of genetic material in single cells and quantitation of cellular subpopulations that have particular genetic aberrations. Comparative genomic hybridization can identify regions of genetic gain and loss in tumor DNA.

Brain Neoplasms↗

PubMed1995

Heterogeneity, polyploidy, aneusomy, and 9p deletion in human glioblastoma multiforme.

The short arm of chromosome 9 is frequently deleted in malignant gliomas. We used locus-specific probes for interferon-A (IFNA) and D9S3 in combination with a chromosome 9 centromeric probe to detect genetic aberrations on a cell-by-cell basis in touch preparations of 30 glioblastomas by fluorescence in situ hybridization. Seven (23%) of 30 tumors had deletions in > 70% of cells; the IFNA locus was deleted in all seven, but the D9S3 locus was deleted in only five of the seven. The latter data confirm that a tumor suppressor gene on 9p relevant to glioblastoma multiforme lies between D9S3 and IFNA. Eleven tumors had deletions in 20-40% of cells, more than three standard deviations above the level in control tissues. The remaining tumors had deletions in < 20% of cells. The seven tumors with the lowest percentage of deleted cells each had more than one genetically abnormal population of cells. In total, 10 cases were of this type (i.e., aneusomic for chromosome 9). Three of these 10 tumors had hybridization patterns consistent with polyploidy.

Adult↗

PubMed1994

Design and testing of novel cytotoxic polyamine analogues.

We designed three polyamine analogues, 1,14-diamino-N5-methyl-5,10- diazatetradecane (5me-4-4-4), 1,14-diamino-N5,N5-dimethyl-5,10-diazatetradecane (Q-Amm-4-4-4), and 1,14-bis-(ethylamino)-N5,N5-dimethyl-5,10-diazatetradecane (BE-Q-Amm-4-4-4), on the basis of computer modeling and physical-chemical studies of polyamine-DNA interactions. These analogues differ from natural polyamines and from one another in the charge distribution on their aliphatic backbone. We found that 10 microM 5me-4-4-4 did not inhibit growth and was not cytotoxic to the human brain tumor cell lines SF-767 and SF-126. The same concentrations of Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 inhibited cell growth and killed more than 90% of each cell type on day 7 of the experiment. BE-Q-Amm-4-4-4 was slightly more toxic than Q-Amm-4-4-4 in both cell lines. All three agents either decreased or completely depleted intracellular putrescine and spermidine. Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 each also lowered spermine. The fact that 5me-4-4-4 was nontoxic but that Q-Amm-4-4-4 was cytotoxic and inhibited growth suggests that the charge distribution along the surface of the aliphatic backbone of polyamines is important in determining growth inhibition and cytotoxicity.

Antineoplastic Agents↗