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Biomedical subjects

A Brinkman

Publications and source records attributed to A Brinkman.

At least 19 recordsLinked to original sources
PubMed2001

Tamoxifen resistance in breast cancer: elucidating mechanisms.

Tamoxifen has been used for the systemic treatment of patients with breast cancer for nearly three decades. Treatment success is primarily dependent on the presence of the estrogen receptor (ER) in the breast carcinoma. While about half of patients with advanced ER-positive disease immediately fail to respond to tamoxifen, in the responding patients the disease ultimately progresses to a resistant phenotype. The possible causes for intrinsic and acquired resistance have been attributed to the pharmacology of tamoxifen, alterations in the structure and function of the ER, the interactions with the tumour environment and genetic alterations in the tumour cells. So far no prominent mechanism leading to resistance has been identified. The recent results of a functional screen for breast cancer antiestrogen resis- tance (BCAR) genes responsible for development of tamoxifen resistance in human breast cancer cells are reviewed. Individual BCAR genes can transform estrogen-dependent breast cancer cells into estrogen-independent and tamoxifen-resistant cells in vitro. Furthermore, high levels of BCAR1/pl30Cas protein in ER-positive primary breast tumours are associated with intrinsic resistance to tamoxifen treatment. These results indicate a prominent role for alternative growth control pathways independent of ER signalling in intrinsic tamoxifen resistance of ER-positive breast carcinomas. Deciphering the differentiation characteristics of normal and malignant breast epithelial cells with respect to proliferation control and regulation of cell death (apoptosis) is essential for understanding therapy response and development of resistance of breast carcinoma.

Antineoplastic Agents, Hormonal↗
PubMed2000

BCAR1/p130Cas expression in untreated and acquired tamoxifen-resistant human breast carcinomas.

High BCAR1/p130Cas expression in primary breast tumour cytosol predicts a poor chance of response recurrent disease to tamoxifen treatment in patients with oestrogen receptor (ER)-positive breast carcinomas. In this study, we assessed whether BCAR1/p130Cas expression is altered during acquisition of anti-oestrogen resistance. BCAR1/p130Cas protein was quantitatively measured by chemiluminescent Western blot analysis in the cytosol of 34 predominantly ER(+) carcinomas that initially responded to primary tamoxifen treatment and subsequently progressed (n = 22 ) or developed during adjuvant tamoxifen treatment (n = 12) and compared to 54 untreated ER(+) human breast carcinomas. We did not detect significant differences in the level of BCAR1/p130Cas protein in untreated and acquired tamoxifen-resistant carcinomas. Our results indicate that in tumour progression towards tamoxifen resistance, increase of BCAR1/p130Cas may be only one of the molecular mechanisms. Thus, high BCAR1/p130Cas protein levels appear to be a hallmark for intrinsic resistance to tamoxifen in breast carcinomas.

Adult↗
PubMed2000

BCAR1, a human homologue of the adapter protein p130Cas, and antiestrogen resistance in breast cancer cells.

BACKGROUND: Treatment of breast cancer with the antiestrogen tamoxifen is effective in approximately one half of the patients with estrogen receptor-positive disease, but tumors recur frequently because of the development of metastases that are resistant to tamoxifen. We have previously shown that mutagenesis of human estrogen-dependent ZR-75-1 breast cancer cells by insertion of a defective retrovirus genome caused the cells to become antiestrogen resistant. In this study, we isolated and characterized the crucial gene at the breast cancer antiestrogen resistance 1 (BCAR1) locus. METHODS/RESULTS: Transfer of the BCAR1 locus from retrovirus-mutated, antiestrogen-resistant cells to estrogen-dependent ZR-75-1 cells by cell fusion conferred an antiestrogen-resistant phenotype on the recipient cells. The complete coding sequence of BCAR1 was isolated by use of exon-trapping and complementary DNA (cDNA) library screening. Sequence analysis of human BCAR1 cDNA predicted a protein of 870 amino acids that was strongly homologous to rat p130Cas-adapter protein. Genomic analysis revealed that BCAR1 consists of seven exons and is located at chromosome 16q23.1. BCAR1 transcripts were detected in multiple human tissues and were similar in size to transcripts produced by retrovirus-mutated ZR-75-1 cells. Transfection of BCAR1 cDNA into ZR-75-1 cells again resulted in sustained cell proliferation in the presence of antiestrogens, confirming that BCAR1 was the responsible gene in the locus. CONCLUSIONS: Overexpression of the BCAR1 gene confers antiestrogen resistance on human ZR-75-1 breast cancer cells. Overexpression of BCAR1 in retrovirus-mutated cells appears to result from activation of the gene's promoter. The isolation and characterization of this gene open new avenues to elucidating mechanisms by which the growth of human breast cancer becomes independent of estrogen.

Amino Acid Sequence↗
PubMed2000

Bcar1/p130Cas protein and primary breast cancer: prognosis and response to tamoxifen treatment.

BACKGROUND: The product of the Bcar1/p130Cas (breast cancer resistance/p130Crk-associated substrate) gene causes resistance to antiestrogen drugs in human breast cancer cells in vitro. To investigate its role in clinical breast cancer, we determined the levels of Bcar1/p130Cas protein in a large series of primary breast carcinomas. METHODS: We measured Bcar1/p130Cas protein in cytosol extracts from 937 primary breast carcinomas by western blot analysis. The levels of Bcar1/p130Cas protein were tested for associations and trends against clinicopathologic and patient characteristics, the lengths of relapse-free survival and overall survival (n = 775), and the efficacy of first-line treatment with tamoxifen for recurrent or metastatic disease (n = 268). RESULTS: Bcar1/p130Cas levels in primary tumors were associated with age/menopausal status and the levels of estrogen receptor and progesterone receptor. In univariate survival analysis, higher Bcar1/p130Cas levels were associated with poor relapse-free survival and overall survival (both two-sided P =.04; log-rank test for trend). In multivariate analysis, a high level of Bcar1/p130Cas was independently associated with poor relapse-free survival and overall survival. The response to tamoxifen therapy in patients with recurrent disease was reduced in patients with primary tumors that expressed high levels of Bcar1/p130Cas. In multivariate analysis for response, Bcar1/p130Cas was independent of classical predictive factors, such as estrogen receptor status, age/menopausal status, disease-free interval, and dominant site of relapse. CONCLUSION: Patients with primary breast tumors expressing a high level of Bcar1/p130Cas protein appear to experience more rapid disease recurrence and have a greater risk of (intrinsic) resistance to tamoxifen therapy. Thus, measurement of Bcar1/p130Cas may provide useful prognostic information for patients with primary or metastatic breast cancer.

Adult↗
PubMed1994

Magical contagion and AIDS risk perception in a college population.

This study examined whether common reactions to AIDS are consistent with operation of the "magical law of contagion," a traditional belief that describes the transfer of properties, whether moral or physical, harmful or beneficial, through contact. Three features of magical contagion, explored in previous work, were re-examined. These features sometimes contrast with microbial contamination as described by modern germ theory. They are: permanence of effects; dose-insensitivity; and potential for effects to act backwards (i.e., from recipient back onto source). A fourth characteristic, previously unaddressed, was also explored: "moral-germ conflation," i.e., the tendency to incompletely distinguish illness from evil. Three hundred and ninety-nine college students completed a survey assessing each feature with regard to AIDS-related scenarios. Also assessed was general AIDS knowledge. Subjects were very well-informed about AIDS, yet a significant subset showed "magical" features of thinking. Consistent with moral-germ conflation, degree of worry about getting AIDS was better predicted by guilt than by risk behaviors and knowledge that they are risky. Implications are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Insulin-like growth-factor-binding protein gene expression and protein production by human tumour cell lines.

The secretion of insulin-like growth-factor-binding proteins (IGFBPs) and expression of the genes encoding IGFBP-1, IGFBP-2 and IGFBP-3 have been studied in a panel of cell lines derived from breast carcinomas, Wilms' tumour, neuroblastoma, retinoblastoma, colon carcinoma, liver adenocarcinoma, Burkitt's lymphoma and a non-small-cell lung carcinoma. All cell lines, with the exception of the Burkitt's lymphoma cell line, secreted IGFBPs, as detected by affinity labelling. A 34-kDa BP was present in the conditioned media of all IGFBP-secreting cell lines, whereas BPs ranging from 18 kDa to 53 kDa were variably secreted. All IGFBP-secreting cell lines expressed the IGFBP-2 gene as determined by Northern blot analysis. The Wilms' tumour, the neuroblastoma and the retinoblastoma cell line expressed the IGFBP-2 gene only. All other cell lines, with the exception of the Burkitt's lymphoma, expressed the IGFBP-2 gene and, in addition, either the IGFBP-1 gene and/or the IGFBP-3 gene. IGFBP-1 gene expression could be detected by reverse transcriptase polymerase chain reaction only. IGFBP-3 gene expression was detected by Northern blot analysis, but transcripts were less abundant than IGFBP-2 mRNAs. These findings indicate that the expression of multiple BP genes and the secretion of BPs may be a common property of tumour cells.

Affinity Labels↗
PubMed1992

Expression of insulinlike growth factor (IGF) and IGF-binding protein genes in human lung tumor cell lines.

BACKGROUND: The presence of multiple, low-molecular-weight, insulinlike growth factor (IGF)-binding proteins in lung tumor cell-conditioned medium and lung cancer patient serum has been recently reported. PURPOSE: To begin to elucidate the genetic basis for these observations, the present study examines the expression by lung tumor cell lines of three IGF-binding protein genes, namely, IGFBP-1, IGFBP-2, and IGFBP-3. Since IGF-binding proteins are thought to modulate the biologic action of the IGFs, the relationship between the expression of IGF-binding protein genes and the genes encoding IGF-I and IGF-II also has been investigated. METHODS: Gene expression was studied in four small-cell lung cancer (SCLC) and three non-small-cell lung cancer (NSCLC) cell lines using Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) for IGFBP-1. RESULTS: IGFBP-1 gene expression was detected by Northern blot analysis in one NSCLC cell line only. However, RT-PCR revealed that the IGFBP-1 gene was expressed in all four SCLC cell lines and in two of the three NSCLC lines. Northern blot analysis of IGFBP-2 gene expression demonstrated that all lung tumor cell lines expressed this gene. A low level of IGFBP-3 gene expression was detected in one SCLC cell line and in all three NSCLC cell lines. All lung tumor cell lines expressed the IGF-II gene as determined by Northern blot analysis. In marked contrast, none of the lines showed evidence of IGF-I gene expression using this method. However, RT-PCR revealed a low level of IGF-I gene expression in one SCLC and one NSCLC cell line only. CONCLUSIONS: These observations indicate 1) that IGF-binding proteins secreted by lung tumors are encoded by at least three different genes; 2) that there may be a close association between IGF-II and IGFBP-2 gene expression, such that, where there is production of IGF-II, IGFBP-2 is the principal BP; and 3) that the IGF-II gene is more widely expressed than the IGF-I gene in human lung tumor cell lines.

Base Sequence↗
PubMed1992

Effects of insulin-like growth factors and growth hormone on the in vitro proliferation of T lymphocytes.

The insulin-like growth factors I and II (IGF-I and IGF-II) promote proliferation and differentiation of many cell types. We report that recombinant IGF-I and IGF-II augment both the lectin- and anti-CD3-induced proliferation of human peripheral blood mononuclear cells (PBMC) at concentrations proportional to their binding affinities. IGF-I and IGF-II also augmented the lectin-induced proliferation of purified T lymphocytes. Effects of IGF-I were found in cultures of T cells vigorously depleted for monocytes and supplemented with saturating concentrations of interleukin-1. The latter results indicate that the effect of IGF-I on the proliferation of T lymphocytes can occur independent of monocytes or monocyte-derived factors.

Carrier Proteins↗
PubMed1992

Cardiac autonomic mechanisms associated with borderline hypertension under varying behavioral demands: evidence for attenuated parasympathetic tone but not for enhanced beta-adrenergic activity.

Elevated blood pressure in psychophysiological studies of borderline hypertension is frequently attributed to the effects of increased sympathetic tone, and with few exceptions, the potential parasympathetic contributions have not been considered. Furthermore, of the investigations that have addressed vagal influences upon blood pressure, most have employed invasive pharmacological assessment of parasympathetic tone. In this study, cardiac parasympathetic and beta-adrenergic influences in borderline hypertension were evaluated noninvasively employing respiratory sinus arrhythmia as a vagal index and preejection period as a sympathetic index of cardiac functioning. Subjects were 30 borderline hypertensive and 23 normotensive males (age range, 24-45 years). The ECG, blood pressure, impedance cardiography, and respiration were measured during two baselines (initial and post-task), a memory-comparison reaction time task, the cold pressor, and CO2-rebreathing. Results indicated tonic differences between groups in all cardiovascular variables across tasks, with the exception of pre-ejection period, which showed no group effects at all. Hypertensives additionally manifested somewhat heightened systolic blood pressure reactivity and attenuated cardiac parasympathetic responsivity to specific tasks. Our findings provide no support for an exaggerated cardiac beta-adrenergic tonic level or reactivity in borderline hypertensives. On the other hand, the consistently lower magnitude of respiratory sinus arrhythmia in our hypertensives suggests that reduced parasympathetic control may be involved in the pathophysiology of hypertension.

Adult↗
PubMed1992

Structural aspects of the IGFBP family.

To date six IGF binding proteins (IGFBP) have been characterized. Analysis of the amino acid sequence reveals that the IGFBPs are clearly distinct but are sharing regions with strong homology. Specifically the hydrophobic cysteine rich N-terminal region and to a lesser extend the C-terminal part are preserved. The alignment of the cysteine molecules is strongly conserved across the 6 IGFBPs. The middle one-third region, where no cysteines are present (except for IGFBP-4) is most divergent. IGFBP-3 and -4 are glycosylated, whereas IGFBP-1 and -2 contain an Arg-Gly-Asp sequence near the carboxyl terminus. Determination of the number of free-SH groups of IGFBP-1 and -3 has revealed that most likely all cysteine residues are involved in disulfide bond formation. All members of the IGFBP family bind IGF-I and IGF-II with about equal affinity. Studies involving deletion mutation and site-directed mutagenesis of IGFBP-1 and -3 have suggested that the three-dimensional structure of the protein plays an important role in IGF binding. However at present it is unclear whether the IGFBPs share one or more specific IGF binding domain. The predominant function of the IGFBPs is to allocate IGF in the various body fluid compartments and tissues and to modulate IGF binding to receptors. For this purpose there exists a very sophisticated control of the routing of circulating IGF both from and to the cell. There is mounting evidence that the structure of the IGFBP proteins plays a key role in the regulation of IGF bioavailability, by modulating its molecular size, capillary membrane permeability, target tissue specificity, cell membrane adherence and IGF affinity.

Amino Acid Sequence↗
PubMed1991

Site-directed mutagenesis of the N-terminal region of IGF binding protein 1; analysis of IGF binding capability.

To define domains involved in IGF binding 60 N-terminal amino acid residues of IGFBP-1 were deleted. This deletion resulted in loss of IGF binding suggesting that the N-terminus may enclose an IGF binding domain. However, most point mutations introduced in this region did not affect IGF binding. In contrast to Cys-34, only substitution of Cys-38 for a tyrosine residue abolished IGF binding. With the determination that all 18 cysteine residues are involved in disulphide bond formation our data suggest that, although not all cysteines contribute to the same extent, the ligand binding site may be spatially organized.

Amino Acid Sequence↗
PubMed1991

IGFBP-1, an insulin like growth factor binding protein, is a cell growth inhibitor.

A novel cell growth inhibitor, IDF45 (inhibitory diffusible factor), was recently purified to apparent homogeneity. It is a bifunctional molecule: able to bind Insulin like growth factor (IGF) and to 100% inhibit DNA synthesis stimulated by serum in fibroblasts. It was of interest to verify whether other members of the IGF-binding protein (IGFBP) family show the same bifunctional growth inhibitory properties. In this paper we show that purified IGFBP-1 derived from amniotic fluid is a cell growth inhibitor. In chick embryo fibroblasts, it inhibited DNA synthesis stimulated by serum. However the stimulation was maximally 60% inhibited and half of the inhibition was observed with 100ng/ml IGFBP-1. So the specific activity of IGFBP-1 is lower than that of IDF45. IGFBP-1 also reversibly prevented the CEF growth. In the same cells IGFBP-1 inhibited DNA synthesis stimulated by IGF-I. We demonstrated that the same protein IGFBP-1 is able to inhibit DNA synthesis stimulated by serum and by IGF-I. The possibility that IGFBP-1 is a bifunctional molecule is discussed.

Amniotic Fluid↗
PubMed1991

Mutations in the C-terminal part of insulin-like growth factor (IGF)-binding protein-1 result in dimer formation and loss of IGF binding capacity.

In an attempt to define domains in insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) that are involved in IGF binding, we subjected the carboxyl end of the coding region of IGFBP-1 cDNA to mutagenesis. Mutant cDNAs were isolated, characterized by sequencing, and cloned in an expression vector under control of the simian virus-40 (SV40) early promoter. The constructs were transfected into COS-1 cells, and the mutant proteins, secreted into the culture medium, were analyzed for IGF binding by ligand blotting. The results obtained show that deletion of the C-terminal 20 amino acids or introduction of frame-shifts in this region resulted in loss of IGF binding and for some mutants in the formation of dimeric IGFBP-1 molecules. These dimers are probably formed when cysteine-226 (Cys-226) is missing, and its putative partner is able to form intermolecular disulfide bonds. Site-directed mutagenesis demonstrated that most of the introduced point mutations in the C-terminal region did not affect IGF binding. Only mutation of Cys-226 to tyrosine completely abolished IGF binding, as did the introduction of a negatively charged amino acid in the vicinity of this residue. Again, dimers were observed, supporting that Cys-226 is essential for the conformation of IGFBP-1. In addition, our data suggest that an IGF-binding domain may be located in the vicinity of the intramolecular disulfide bond formed by Cys-226 and its putative partner.

Amino Acid Sequence↗
PubMed1988

Organization of the gene encoding the insulin-like growth factor binding protein IBP-1.

Human genomic clones encompassing the tissue specific expressed gene IBP-1, an insulin-like growth factor binding protein were isolated and characterized. The gene is organized in four exons and spans 5.9 kb. S1 nuclease analysis determined a single transcription start site. The first exon and 5' flanking region are highly GC rich and located in a CpG island. The CpG island enclose the CAAT box, the TATA box, the transcription start site and a potential SP1 transcription factor binding site. The presumptive promoter region is characteristic for genes expressed in a tissue specific fashion. All signals required for cleavage/polyadenylation are located within exon IV, predicting a mRNA of 1.5 kb which is consistent with the size seen on RNA blots.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of a cDNA encoding the low molecular weight insulin-like growth factor binding protein (IBP-1).

IGF-I and IGF-II are growth-stimulating peptides with strong mitogenic properties. These polypeptide growth factors circulate in serum bound to specific binding proteins. We report the cloning and complete sequence of a cDNA encoding a low mol. wt IGF-binding protein from a human placenta cDNA library. We propose the designation IGF-binding protein 1 (IBP-1) for the gene and corresponding protein. Expression of the cDNA encoding IBP-1 in COS cells resulted in the synthesis of a 30-kd protein which binds IGF-I and is immunologically indistinguishable from the IGF-binding protein isolated from amniotic fluid or human serum. Northern blotting analysis demonstrated that expression of the IBP-1 gene is highly tissue specific and limited to placental membranes and fetal liver suggesting a rigid control. The IBP-1 gene is a single copy gene, located on chromosome 7. The results obtained suggest that most, if not all, lower mol. wt IGF-binding proteins originate from this gene.

Amino Acid Sequence↗
PubMed1984

Analysis of the methylation-regulated Mu mom transcript.

The Mu mom gene is transcribed only when DNA is methylated by Dam; this methylation dependence is determined by a region upstream of the gene containing three Dam-methylation sites. The mom operon contains two open reading frames, one of which codes for Mom. Experiments were done to find a function for the other reading frame; it is shown that it does not affect phage immunity or the regulation of mom expression. Deletion mapping and S1 nuclease mapping experiments identify the +1 of the mom mRNA: it is 45 bp downstream of the three Dam-methylation sites. Possible models for the short-distance transcription regulation by methylation are discussed. The mom promoter is surrounded by sequences that are shown to be homologous to the oriC region of E. coli. This suggests a relation between replication and methylation-dependent mom expression. It is shown however, that replication is not essential for mom expression.

Bacteriophage mu↗
PubMed1983

DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems.

The gene product of bacteriophage Mu gin catalyzes a 3,000-base-pair inversion in the DNA of the phage, thus changing its host range. In some strains of Escherichia coli there is a function that can complement Mu gin mutations. This function (pin) was cloned and shown to catalyze an inversion of 1,800 base pairs in the adjacent E. coli DNA (P region). pin- derivatives carry the P region frozen in the (+) or (-) orientation. The function of the switch is not yet clear. The sequences of gin and pin were determined; they exhibit 70% homology. The sequences around the recombination sites of Gin and Pin are also largely homologous; a consensus sequence is derived for the recombination sites of Gin and Pin, and of Hin in Salmonella typhimurium. The amino acid sequences of Gin, Pin, Hin, and TnpR are compared, and the evolutionary relationship between these prokaryotic site-specific recombination systems is discussed.

Amino Acid Sequence↗