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A Neubauer

Publications and source records attributed to A Neubauer.

At least 145 records · Page 8Linked to original sources
PubMed1994

Detection of allelic loss within the beta 1-interferon gene in childhood acute lymphoblastic leukemia using differential PCR.

Deletion of the short arm of chromosome 9p involving the beta 1-interferon (IFN) gene has been implicated in the process of malignant transformation in lymphomas and acute lymphoblastic leukemias. Since cytogenetic analysis is frequently unsuccessful in clinical samples, we used a recently described differential PCR technique to detect losses within the beta 1-IFN gene in 86 acute leukemias. Using differential PCR, no beta 1-IFN deletion was detected in 44 acute myeloid leukemia (AML) and eight control samples. However, five of 42 acute lymphoblastic leukemia (ALL) probes (12%) exhibited loss of the beta 1-IFN gene (three common ALL, two T-ALL). Cytogenetic analysis was performed independently in three of these five cases and revealed abnormalities of chromosome 9p in two samples. Two of five T-ALL cases exhibited a loss within the beta 1-IFN gene, compared with 3/29 c-ALLs, suggesting a predominance of IFN gene loss in T-ALLs. These data indicate that PCR can be used for rapid detection of gene dosage phenomena in clinical leukemia samples.

Acute Disease↗
PubMed1994

Human peritoneal mesothelial cell prostaglandin synthesis: induction of cyclooxygenase mRNA by peritoneal macrophage-derived cytokines.

Increasing evidence suggests that the mesothelial cell contributes to the control of inflammation in both the normal and inflamed peritoneal cavity. The present study examines the regulation of prostaglandin production by human peritoneal mesothelial cells (HPMC) following stimulation with peritoneal macrophage-conditioned medium and the cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). IL-1 beta and TNF-alpha stimulated significant release of prostaglandin above background levels in a time and dose dependent manner. Stimulation of HPMC with IL-1 beta (500 pg/ml) or TNF-alpha (100 pg/ml) for 24 hours resulted in the release of 24.5 +/- 4.3 (N = 11) (z = 3.40, P < 0.001 vs. control) and 19.4 +/- 4.5 (N = 10; z = 3.29, P < 0.001 vs. control) pg 6-keto-PGF1 a/micrograms cellular protein, respectively. Pretreatment of HPMC with dexamethasone (10(-6) to 10(-9) M) inhibited both constitutive and cytokine stimulated prostaglandin synthesis in a dose dependent manner. Both PMø-CM and PMø-S.epiCM stimulated 6-keto-PGF1 alpha and PGE2 synthesis by HPMC in a time and dose dependent manner (PMø-S.epiCM >> PMø-CM). Co-incubation of HPMC with PMø-S.epiCM in the presence of anti-IL-1 beta and/or anti-TNF-alpha antibody, interleukin-1 receptor antagonist or soluble TNF receptor (TNF p75) significantly reduced the capacity of these supernatants to stimulate prostaglandin synthesis. Exposure of HPMC to cytokines or PMø-S.epiCM resulted in the time dependent increase in the levels of both Cox-1 and Cox-2 mRNA as assessed by RT/PCR analysis with the greatest increase being seen for Cox-2. These data demonstrate specific stimulation of eicosanoid metabolism in HPMC by peritoneal macrophage derived cytokines, indicating the possible importance of these mediators in the activation of intraperitoneal prostaglandin synthesis. HPMC prostaglandins might act as important pro/anti-inflammatory mediators contributing to a cytokine network in the peritoneal cavity during CAPD peritonitis.

Base Sequence↗
PubMed1994

Mutations in the ras proto-oncogenes in patients with myelodysplastic syndromes.

Activation of the N- and K-ras proto-oncogenes is the most common molecular abnormality in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). In retrospective studies, approximately 3-36% of MDS patients were reported to harbor a mutated ras proto-oncogene, with some series suggesting the presence of ras-mutations are associated with progressive disease and a poor prognosis. Since hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) are currently used for therapy in MDS but may stimulate the proliferation of leukemic cells, we assessed the frequency and significance of ras mutations in 27 MDS patients, 15 of whom underwent G-CSF therapy. Patients were analyzed for the presence of mutations in codons 12, 13, and 61 of the N- and K-ras proto-oncogenes. Only three patients (11%, two refractory anemia with excess of blasts (RAEB), one RAEB in transformation (RAEB-T)) harbored activated ras oncogenes with the mutations localized in N-ras codons 12 and 61. Patients were followed for periods of up to 4 years or until death supervened. Patients exhibiting ras mutations were no more likely to develop AML compared to ras-negative patients (1/3 vs. 10/24) or to have decreased survival (p = 0.64). These data indicate that, in this group of MDS patients, ras mutations do not appear to correlate with a poor prognosis, and do not adversely interact with exogenously administered G-CSF.

Acute Disease↗
PubMed1994

In-vitro biocompatibility of alternative CAPD fluids; comparison of bicarbonate-buffered and glucose-polymer-based solutions.

Evidence is accumulating that conventional dialysis fluids for CAPD are incompatible with peritoneal host defence. We therefore investigated the effect of alternative CAPD fluids on mononuclear leukocyte (PBMC) viability and cytokine production in vitro. Fluids tested were bicarbonate-buffered solutions containing 1.5% or 4.25% glucose, 7.5% glucose polymer dialysis fluid (GPDF), and conventional 1.5% glucose fluid (G1.5%). PBMC were stimulated (2 h, 37 degrees C) in the different test fluids with a clinical isolate of Staphylococcus epidermidis or Escherichia coli lipopolysaccharide. The cytokines TNF alpha and IL-6 in PBMC supernatants were measured by specific enzyme immunoassays. Induction of cytokine messenger RNA was evaluated by reverse transcription-polymerase chain reaction. Conventional G1.5% (pH 5.5) inhibited cytokine release from activated PBMC by > 95%, whereas cell responses in low-glucose bicarbonate fluid were not significantly reduced. In contrast, high-glucose bicarbonate fluid exerted > 80% inhibition despite its neutral pH. GPDF was inhibitory at its initial low pH, whereas cytokine release was restored following pH neutralization. Cytokine mRNA expression was suppressed by conventional G1.5% fluid and by high-glucose bicarbonate fluid. These data indicate that pH neutralization leads to a substantial improvement of dialysis fluid biocompatibility; however, hyperosmolality and/or high glucose content inhibit cell responsiveness even at normal pH. Replacement of glucose by glucose polymer might prove beneficial provided that the initial low pH is neutralized.

Actins↗
PubMed1994

Ras point mutations occur in acute myeloid leukemia with illegitimate T-cell receptor delta gene rearrangement.

Mutations in the ras proto-oncogenes are the most frequently observed molecular alteration in acute myeloid leukemia (AML). Whether ras mutations occur as late or relatively early events in the multistep process of myeloid transformation, remains an open question. We previously described illegitimate T-cell receptor (TCR)-delta gene rearrangements in a subset of AML. These recombinations were detected in 9 out of 100 de novo AML cases. Southern blot analysis suggested the presence of these recombinations in the vast majority of AML cells and thus could be used as clonal markers. In order to more accurately define the role of ras proto-oncogene mutations in the multistep process of malignant transformation in myeloid leukemias, we performed single strand conformation polymorphism (SSCP) assays, slot blot and direct sequencing analysis on these nine cases with illegitimate TCR delta gene rearrangements. Ras proto-oncogene mutations were found in three of nine cases. Interestingly, SSCP, slot blot and sequencing suggested the presence of the respective mutations in most of the leukemic cells. Thus, ras mutations presumably occurred early in the process of transformation in these three cases.

Adult↗
PubMed1993

Detection of p53 mutations using nonradioactive SSCP analysis: p53 is not frequently mutated in myelodysplastic syndromes (MDS).

p53 is one of the most frequently mutated genes in human cancers. Since p53 has been implicated in lymphatic and some myeloid leukemias, such as the blastic phase of chronic myelogenous leukemia, we sought to address the role of p53 gene mutations within exons 4-9 in myelodysplastic syndromes (MDS), a myeloid preleukemic condition. In order to avoid the potential hazard of using radioactive single-strand conformation analysis (SSCP), we used a nonradioactive SSCP method based on the silver stain of small minigels. In cell lines with known point mutations of the p53 gene, aberrant migrating bands were found. Serial dilutions indicated a sensitivity comparable to radioactive methods. Furthermore, a common polymorphism within the 4th exon of the p53 gene was easily detected. However, of 17 primary samples from patients with MDS, none harbored a p53 gene mutation. We conclude that this nonradioactive method can easily be used to screen for p53-gene mutations, and that p53-gene mutations do not play a major role in the pathogenesis of MDS.

Alleles↗
PubMed1993

Molecular alterations in a patient with Turcot's syndrome.

Cells of a patient with Turcot's syndrome and of her parents were evaluated for the presence of molecular alterations in the p53 and the Ki-ras gene. Deletions on chromosome 17p, overexpression and point mutations of the p53 gene as well as mutations of the Ki-ras gene were detected in primary and metastatic tumour but not in the germline of the patient nor in her parents.

Adenocarcinoma↗
PubMed1993

Altered expression of the retinoblastoma susceptibility gene in chronic lymphocytic leukaemia.

The pathogenesis of chronic lymphocytic leukaemia (CLL) is unknown. One of the most frequent cytogenetic abnormalities in CLL is a deletion within the long arm of chromosome 13, the region to which the retinoblastoma (Rb) gene has been mapped. Lack of Rb expression has been linked to the carcinogenic process in many human tumours. We therefore sought to investigate the role of Rb gene inactivation in CLL using differential polymerase chain reaction on reverse transcribed RNA. The result of the PCR was quantitated using HPLC. 5/39 patients revealed a lack or significantly impaired expression of the Rb gene upon differential PCR analysis. In addition, immunocytochemical studies were performed using the Rb-specific monoclonal antibody PMG245. 10/56 patients showed a weak or absent expression upon immunocytochemical analysis compared to monocytes or granulocytes. The samples lacking Rb were from both early and late stage CLL. Our results indicate that inactivation of the Rb protein occurs in a fraction of CLL cases and can be found in early and late stages of the disease.

Aged↗
PubMed1993

The chronic myelocytic cell line K 562 contains minor (m) as well as major (M) ber/abl fusion mRNAs.

By searching for additional chimeric bcr/abl transcripts in K 562 cells characterized by major (M) bcr/abl fusions, a new mRNA, a minor (m) bcr/abl transcript, was detected. A practical implication of this finding is that the K 562 cell line can be used as positive control for the detection by the polymerase chain reaction of both types of transcripts for the diagnosis of Philadelphia chromosome associated leukemias.

Base Sequence↗
PubMed1993

Genetic alterations in the p53 gene in the blast crisis of chronic myelogenous leukemia: analysis by polymerase chain reaction based techniques.

Rearrangements of the c-abl protooncogene and the bcr-gene are found in > 90% of patients in chronic phase of chronic myelogenous leukemia (CML). The molecular events leading to blast crisis, however, have not been well characterized. Gross alterations of the p53 gene have been detected in 30% of patients with blast crisis. Since point mutations in the p53 gene appear to be important in the process of transformation in many epithelial tumors, we looked for these mutations in the critical regions of the p53 gene (exons 4, 5, 6, 7, and 8). We used the polymerase chain reaction (PCR), direct sequencing, differential PCR, and single strand conformation polymorphism (SSCP) analysis to detect mutations of the p53 gene in samples from 21 patients with CML blast crisis. Two of 21 patients exhibited an intragenic deletion or rearrangement in p53. In addition, these patients were homozygous for the mutant p53 allele. No mutations were found in the p53 gene of the remaining 19 patients. However, sequencing of the CML blast crisis cell line, K562, revealed an insertion of a C at base position 956 within the fifth exon, causing a frame shift mutation and an early translational stop at codon 148. We conclude that, in contrast to solid tumors, mutations in exons 4-8 of p53 are not frequently seen in primary samples from CML blast crisis. However, deletions and/or rearrangements within the p53 gene do occur and may contribute to the progression from chronic phase to blast crisis in a limited number of patients with CML.

Base Sequence↗
PubMed1992

ras oncogene activation and occupational exposures in acute myeloid leukemia.

BACKGROUND: Epidemiologic studies of acute myeloid leukemias (AMLs) show small increases in risk of disease associated with certain occupations and chemical exposures. PURPOSE: This study was designed to determine whether the presence of mutationally activated ras oncogenes in AML are associated with occupational and chemical exposures. METHODS: We interviewed 62 patients with newly diagnosed AML (or their next-of-kin), all of whom were enrolled in a national multicenter clinical trial, and 630 healthy control subjects. DNA extracted from patients' pretreatment bone marrow samples was amplified by using the polymerase chain reaction and probed with allele-specific oligonucleotides for activating point mutations at the 12th, 13th, and 61st codons of three protooncogenes: H-ras (also known as HRAS), K-ras (also known as KRAS2), and N-ras (also known as NRAS). RESULTS: Patients with ras mutation-positive AML had a higher frequency (six of 10 patients) of working 5 or more years in an a priori high-risk occupation than did patients with ras mutation-negative AML (eight of 52; odds ratio [OR] = 6.8; 95% confidence interval [CI] = 1.3-36). Patients with ras mutation-positive AML were more likely than patients with ras mutation-negative AML to have breathed chemical vapor on the job (OR = 9.1; 95% CI = 1.3-64) or to have had skin contact with chemicals (OR = 6.9; 95% CI = 1.3-37). When ras-positive patients were compared with healthy control subjects, the ORs for occupation and occupational exposures remained elevated, while patients with ras mutation-negative AML showed no increased risk when compared with control subjects. CONCLUSION: Activation of ras proto-oncogenes may identify an etiologic subgroup of AML caused by occupation and chemical exposure. IMPLICATION: Disease etiology may be better understood if epidemiologic measures of exposure are integrated with molecular assays of the genetic defects responsible for cancer initiation and promotion.

Acute Disease↗
PubMed1992

PCR-determined expression of the MDR1 gene in chronic lymphocytic leukemia.

To determine the role the multiple drug-resistance (MDR 1) gene plays in chronic lymphocytic leukemia (CLL), we measured the expression of the MDR 1 gene in 30 patients with this disease. A rapid, highly sensitive, and nonradioactive technique based on the polymerase chain reaction (PCR) was used for that purpose. In this technique, called differential PCR, the target (MDR 1) and a reference gene (beta 2-microglobulin) are co-amplified by PCR from random hexamer-primed cDNA in the same reaction vessel. The level of target gene expression is reflected in the ratio between the intensities of the two resulting PCR product bands, as measured by high-performance liquid chromatography (HPLC). MDR 1 gene expression was detectable in 29/30 (97%) patients with CLL, with a median expression level of 0.36 U (human placenta = 1 U). There was no correlation between expression of the MDR 1 gene and clinical stage, time from diagnosis, absolute lymphocyte count, several lymphocyte surface markers, or prior treatment in the patients analyzed. Immunocytochemical studies of the same material using the monoclonal antibody C219 showed a very low or undetectable expression of the P-glycoprotein in the lymphocytes of all patients studied, whereas granulocytes were significantly more immunoreactive. We conclude that the level of expression of the MDR 1 gene in CLL is generally low, that the removal of granulocytes is important in studies of expression of MDR 1 mRNA in CLL, and that differential PCR provides a rapid and reliable method for quantifying the amount of a specific mRNA, even in very small samples of total RNA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Human papillomavirus types 16 and 18 are not involved in human prostate carcinogenesis: analysis of archival human prostate cancer specimens by differential polymerase chain reaction.

Human papilloma viruses (HPV) have been implicated in the pathogenesis of a variety of malignancies, especially in carcinomas of the female genital tract. Recently, based on observations using the polymerase chain reaction amplification assay, HPV types 16 and 18 specific DNA sequences have been detected in prostate cancer specimens obtained by transurethral resection. Since HPV types 16 and 18 have been shown to possess oncogenic potential, an association between HPV infection and prostatic carcinoma has been suggested. In order to exclude potentially HPV-colonized urethral mucosa from analysis and restrict our study to predominantly malignant tissue, cancerous areas from a series of 30 paraffin-embedded prostate adenocarcinomas were microdissected and analyzed for the presence of HPV 16 or HPV 18 specific sequences by a modification of PCR (D-PCR) and Southern blot analysis. Despite the high sensitivity of our analytical technique, we found no evidence of HPV-DNA of either type in any of the 30 primary prostate cancers. In contrast, both HPV 16 (2/8 specimens) and HPV 18 (2/8 specimens) DNA was detected in randomly chosen cervical carcinomas using the D-PCR methodology. Our data would indicate that the oncogenic HPV-types 16 and 18 are unlikely effectors of prostate carcinogenesis.

Adenocarcinoma↗
PubMed1992

Alterations of the P53 gene are associated with the progression of a human prostate carcinoma.

P53 is a tumor suppressor gene that has been implicated in the molecular genetics of many human malignancies. Nucleotide alterations, most commonly single point mutations, have been shown not only to abrogate the p53 suppressor function but also to contribute to the transformed phenotype. We report the detection of a p53 gene mutation in clinical specimens of a patient with relapsing prostate adenocarcinoma 14 years after definitive external beam radiation. The techniques of single strand conformation polymorphism analysis and direct sequencing of polymerase chain reaction generated products were used for this study. Analysis of tissue from different locations of the primary tumor revealed intratumoral molecular heterogeneity; the mutation was absent in 1 area but present in another. Tumor from a regional lymph node metastasis harbored the identical p53 mutation. Furthermore, an additional genetic alteration, an allelic loss on chromosome 17p but not including the p53 gene, was observed only in the metastatic tissue. These observations in clinical specimens of primary and metastatic sites provide evidence for the association of the p53 gene in the progression of human prostate carcinoma.

Adenocarcinoma↗
PubMed1992

Rearrangements of T-cell receptor delta, gamma and beta genes in acute myeloid leukemia coexpressing T-lymphoid features.

In order to investigate the role of T-cell receptor (TcR)-delta and TcR-gamma gene rearrangements and/or deletions in acute myeloid leukemia (AML) coexpressing T-cell-associated antigens (i.e. CD2 and/or CD4 and/or CD7), we examined blasts from a selected group of 56 AML cases (25 children, 31 adults) coexpressing either of these antigens without cytoplasmic CD3 expression. Forty-four typical AML cases (7 children, 37 adults) without T-cell associated antigens were further studied as controls. Germline configuration of the TcR-delta gene was observed in 91 out of the total of 100 AML cases investigated. Eight of nine cases with rearranged or deleted TcR-delta genes coexpressed T-cell-associated antigens. Blast cells of 7/9 cases were classified as FAB M1, two as FAB M2. In six of these cases TcR-gamma gene rearrangements were also detected. TcR-delta alterations were predominantly found in children whose blasts coexpressed T-lymphoid associated antigens (6/25, 24%), but were rarely detected in adult AML with or without coexpression of T-cell antigens (2/31 and 0/37, respectively).

Acute Disease↗