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Biomedical subjects

U Brinkmann

Publications and source records attributed to U Brinkmann.

At least 37 records · Page 2Linked to original sources
PubMed2000

How to manage individualized drug therapy: application of pharmacogenetic knowledge of drug metabolism and transport.

Significant fractions of health budgets must be spent for treatment of drug side effects and for inefficient drug therapy. Hereditary variants in drug metabolizing enzymes, drug transporters, and drug targets are important determinants of drug response and toxicity and may therefore aid in selection and dosage of drugs. Today there is extensive knowledge of genetic polymorphisms of cytochrome P450 (CYP) enzymes 2A6, 2C9, 2C19, and 2D6; of phase-2 enzymes such as thiopurine S-methyltransferase; and more recently of drug transporters such as the MDR-1 gene-product P-glycoprotein, affecting a significant share of currently used drugs. However, application of pharmacogenetic knowledge to clinical routine is limited in current practice. To promote the application of pharmacogenetic knowledge in clinical routine, research on genotype-based dose adjustments is still necessary - as is the promotion of faster and cheaper genotype analyses. Furthermore, the benefits of CYP genotype-directed drug therapy should be evaluated in properly designed prospective studies. Once such steps have been successfully taken, drug therapy could well become more prevention-directed and patient-tailored than it is possible today, replacing the current "one drug in one dose for one disease" strategy by a more individualized approach.

Cytochrome P-450 Enzyme System↗
PubMed1999

High expression of a specific T-cell receptor gamma transcript in epithelial cells of the prostate.

We have identified expression of T-cell receptor gamma chain (TCRgamma) mRNA in human prostate and have shown that it originates from epithelial cells of the prostate and not from infiltrating T-lymphocytes. In contrast, the T-cell receptor delta chain (TCRdelta) gene is silent in human prostate. The major TCRgamma transcript in prostate has a different size than the transcript expressed in thymus, spleen, and blood leukocytes. It is expressed in normal prostate epithelium, adenocarcinoma of the prostate, and the prostatic adenocarcinoma cell line LNCaP. The RNA originates from an unrearranged TCRgamma locus, and it is initiated within the intronic sequence directly upstream of the Jgamma1.2 gene segment. The prostate-specific TCRgamma transcript consists of the Jgamma1.2 and Cgamma1 gene segments, and it has an untranslated sequence including a polyadenylation signal and poly(A) sequence at the 3'end. The finding that prostate epithelial cells express a high level of a transcript from a gene that was thought to by exclusively expressed by T-lymphocytes is highly unexpected.

Amino Acid Sequence↗
PubMed1999

Tissue-specific alternative splicing of the CSE1L/CAS (cellular apoptosis susceptibility) gene.

CSE1L/CAS (CAS) is a nuclear transport factor that plays a role in proliferation and apoptosis. The CAS gene consists of 25 exons. mRNA homologous over its entire length to the yeast homologue CSE1 is the predominant transcript in proliferating tissues. Additional mRNAs are generated by alternative splicing in a tissue-specific manner. An extended 3'-end is found in fetal and adult brain. A mRNA containing the 5'-end of CAS up to position 690 and an alternative 3'-end is expressed in trachea and encodes a truncated Ran-binding domain. Fetal liver expresses a mRNA with deletions of a central portion of CAS and additional sequences encoded by the last intron. SW480 colon cancer cells express another approximately 1500-base mRNA. Western blot analyses of various human tissues and immunohistology of mouse embryos show a correlation of CAS transcripts and CAS protein in different tissues. CAS isoforms may control nuclear transport of tissue-specific proteins.

Alternative Splicing↗
PubMed1999

Apoptosis induced by Pseudomonas exotoxin: a sensitive and rapid marker for gene delivery in vivo.

The rapid progress in gene therapy has expanded our ability to alter genetic structure, necessitating the development of methods for detecting the activity of new vectors. The central concept of a reporter gene is simple: it is a defined nucleotide sequence, which when introduced into a biological system, yields a readily measurable phenotype on expression. This provides a convenient parameter that is correlated to the molecular events associated with genetic expression. In this study we demonstrate that Pseudomonas exotoxin A (PE) can serve as a sensitive reporter gene to detect gene expression in single cells of mouse lung on cationic liposome delivery of PE-encoding DNA in vivo. Furthermore, we show that PE expression can be detected as early as 2 hr after systemic gene delivery in lungs of recipient mice. We compared PE with the widely used beta-galactosidase gene for this purpose. PE induces apoptosis that can be detected by TdT end labeling of DNA fragments (TUNEL assay) Since few expressed PE molecules are necessary to trigger the apoptosis cascade, the minimal amount of PE-encoding plasmid DNA needed for detection of apoptotic cells after systemic delivery was 0.1 microg per animal compared with at least 1 microg for the beta-galactosidase-encoding plasmid DNA. The maximum number of apoptotic cells detected in lungs was about 15-20 times higher than the maximum number of beta-galactosidase-positive cells. Specificity of apoptosis due to PE expression on delivery of the PE-encoding plasmid was shown by prevention of the apoptotic cascade by the caspase inhibitor Z-VAD-fmk.

ADP Ribose Transferases↗
PubMed1999

Novel genes in the PAGE and GAGE family of tumor antigens found by homology walking in the dbEST database.

We have developed a computer-based screening strategy to search the dbEST database to find differentiation antigens that are expressed by cancers arising in nonessential normal tissues such as prostate, breast, and ovary (G. Vasmatzis et al., Proc. Natl. Acad. Sci. USA, 95: 300-304, 1998). Here, we report the identification of three new members of the GAGE/ PAGE family, termed XAGEs. XAGE-1 and XAGE-2 are expressed in Ewing's sarcoma, rhabdomyosarcoma, a breast cancer, and a germ cell tumor. We also describe the relationship of the XAGEs to the GAGE/ PAGE family. XAGE-1 and XAGE-2 should be evaluated as possible targets for vaccine-based therapies of cancer.

Amino Acid Sequence↗
PubMed1999

Expression of the proliferation and apoptosis-associated CAS protein in benign and malignant cutaneous melanocytic lesions.

We have examined the expression of the cellular apoptosis susceptibility protein, a nuclear transport factor that plays a role in apoptosis and cell proliferation, in benign and malignant melanocytic lesions. Tissue samples of 55 formalin-fixed, paraffin-embedded melanoma (primary n=32, metastatic n=23) and of 27 control cases (junctional dermal, compound, Spitz, Reed, blue nevi, balloon-cell nevus, lentigo maligna) were analyzed by immunohistochemistry with anti-cellular apoptosis susceptibility antibodies. The percentage of cellular apoptosis susceptibility-positive cells as well as the intensity on a four-point scale was evaluated. In normal skin, expression of cellular apoptosis susceptibility was primarily found in the basal cell layer of the epidermis. Benign melanocytic lesions that stained positive for cellular apoptosis susceptibility (13 of 27) showed a homogeneously distributed staining pattern with a mean of 5+/-12% cellular apoptosis susceptibility positive cells. Five out of 7 lentigo maligna melanoma, 11 out of 12 superficial spreading melanoma and all acrolentiginous (n=7) and nodular (n=6) melanoma showed immunoreactivity of medium (++) to high ( ) intensity. Vertical growth phases of primary cutaneous melanoma stained stronger than horizontally growing cell clusters. All metastases (n= 23) stained strongly positive, the staining pattern being inhomogeneous. Cellular apoptosis susceptibility detection in clinical stages according to UICC showed an increase from 43+/-34% cellular apoptosis susceptibility positive cells in stage I, to 53+/-26% in stage II, 68+/-24% in stage III and 72+/-24% in stage IV, respectively. Because the expression of cellular apoptosis susceptibility correlates predominantly with advanced stages of melanoma, staining with anti-cellular apoptosis susceptibility antibodies may be useful for diagnosis of melanoma and possibly as an immunohistochemical prognostic factor in cutaneous melanocytic lesions.

Apoptosis↗
PubMed1998

Role of caspases in immunotoxin-induced apoptosis of cancer cells.

Immunotoxins composed of antibodies linked to plant or bacterial toxins are being evaluated in the treatment of cancer. It is known that the toxin moieties of immunotoxins, including Pseudomonasexotoxin A (PE), diphtheria toxin, and ricin, are capable of inducing apoptosis. Since the efficiency of induction of apoptosis and the apoptosis pathway may have direct effects on the therapeutic usefulness of immunotoxins, we have studied how B3(Fv)-PE38, a genetically engineered immunotoxin in which the Fv fragment of an antibody is fused to a mutated form of PE, induces apoptosis of the MCF-7 breast cancer cell line. We show for the first time that a PE-containing immunotoxin activates ICE/ced-3 proteases, now termed caspases, and causes characteristic cleavage of the "death substrate" poly(ADP)-ribose polymerase (PARP) to an 89 kDa fragment with a time course of cleavage comparable to that induced by TNFalpha. Also the fluorescent substrate, DEVD-AFC, is cleaved 2-4-fold more rapidly by lysates from B3(Fv)-PE38 treated MCF-7 cells than untreated control cells, suggesting that a CPP32-like caspase is involved in B3(Fv)-PE38-mediated apoptosis. B3(Fv)-PE38-induced PARP cleavage is inhibited by several protease inhibitors known to inhibit caspases (zVAD-fmk, zDEVD-fmk, zIETD-fmk) as well as by overexpression of Bcl-2 providing additional evidence for caspase involvement. zVAD-fmk, a broad spectrum inhibitor of most mammalian caspases, prevents the early morphological changes and loss of cell membrane integrity produced by B3(Fv)-PE38, but not its ability to inhibit protein synthesis, arrest cell growth, and subsequently kill cells. Despite inhibition of apoptosis, the immunotoxin is still capable of selective cell killing, which indicates that B3(Fv)-PE38 kills cells by two mechanisms: one requires caspase activation, and the other is due to the arrest of protein synthesis caused by inactivation of elongation factor 2. The fact that an immunotoxin can specifically kill tumor cells without the need of inducing apoptosis makes such agents especially valuable for the treatment of cancers that are protected against apoptosis, e.g., by overexpression of Bcl-2.

ADP Ribose Transferases↗
PubMed1998

The hCSE1/CAS protein is phosphorylated by HeLa extracts and MEK-1: MEK-1 phosphorylation may modulate the intracellular localization of CAS.

hCSE1/CAS (CAS), the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation and apoptosis. A MEK-1 phosphorylation sequence in CAS raises the possibility that MEK-phosphorylation regulates the function of CAS. CAS protein from cell extracts shows covalent charge modifications; one of these charge variants contains phosphotyrosine. CAS protein can be captured from cell extracts by immobilized anti-phosphotyrosine antibodies. We have produced recombinant protein fragments containing the N-terminal or central portion of CAS and found that the N-terminal fragment, which contains a putative MEK phosphorylation site, is phosphorylated by the HeLa extracts and MEK-1. Treatment of cells with an inhibitor of MEK-1 phosphorylation in vivo changes the intracellular localization of CAS from predominantly cytoplasmic to nuclear. This suggests that a function of CAS in nuclear transport may be regulated by phosphorylation.

Apoptosis↗
PubMed1998

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus.

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

Amino Acid Sequence↗
PubMed1998

A bivalent disulfide-stabilized Fv with improved antigen binding to erbB2.

We have used protein engineering to generate a stable bivalent Fv molecule of the anti-erbB2 monoclonal antibody e23. The VH and VL domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a flexible 15 amino acid residue (Gly4-Ser)3 linker. The e23 (dsFv)2 molecule is fused to a truncated form of Pseudomonas exotoxin to generate a bivalent disulfide-stabilized, (dsFv)2, immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro and purified to about 95% purity. Binding studies demonstrated that the (dsFv)2 molecule has a much higher affinity for erbB2 than a monovalent dsFv molecule and a similar binding affinity as the parental antibody e23. The (dsFv)2 immunotoxin was 5 to 20-fold more cytotoxic to two e23 antigen-positive cell lines than the monovalent dsFv immunotoxin. The bivalent dsFv molecule is very stable, retaining 94% of its activity after a 24 hours incubation in human serum at 37 degreesC. Two other molecules with shorter linkers five and ten amino acid residues in length were produced and showed similar activities as the molecule containing a 15 amino acid residue linker. The bivalency, stability and the relative ease of purification makes these e23 (dsFv)2 molecules valuable reagents for cancer immunotherapy and diagnosis.

ADP Ribose Transferases↗
PubMed1998

Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis.

A procedure is described to discover genes that are specifically expressed in human prostate. The procedure involves searching the expressed sequence tag (EST) database for genes that have many related EST sequences from human prostate cDNA libraries but none or few from nonprostate human libraries. The selected candidate EST clones were tested by RNA dot blots to examine tissue specificity and by Northern blots to examine the transcript size of the corresponding mRNA. The computer analysis identified 15 promising genes that were previously unidentified. When seven of these were examined in an RNA hybridization experiment, three were found to be prostate specific. The genes identified could be useful in the targeted therapy of prostate cancer. The procedure can easily be applied to discover genes specifically expressed in other organs or tumors.

Cloning, Molecular↗
PubMed1997

Antisense inhibition of CAS, the human homologue of the yeast chromosome segregation gene CSE1, interferes with mitosis in HeLa cells.

We have analyzed the effects on HeLa cells of reduction of the CAS protein, the human homologue to yeast chromosome segregation protein CSE1. Expression of CAS antisense cDNA decreases the amount of CAS protein in HeLa cells and perturbs progression from G2 (retards transition from G2) to G1 in the cell cycle. Increased levels of cyclin B in CAS antisense transfected cells correlated with an arrest in G2 phase or mitosis. This arrest upon CAS attenuation is consistent with observations that yeast with CSE1 mutations are defective in mitosis and cyclin B degradation.

Cellular Apoptosis Susceptibility Protein↗
PubMed1997

Cloning and expression of the recombinant FAb fragment of monoclonal antibody K1 that reacts with mesothelin present on mesotheliomas and ovarian cancers.

The monoclonal antibody (MAb) K1 recognizes an approximate 40 kDa glycoprotein, mesothelin, that is present on the surface of human mesothelial cells, mesotheliomas and ovarian cancers. We have cloned the cDNAs encoding the variable regions of MAb K1 and constructed plasmids for expression of recombinant K1(FAb). Recombinant FAb was produced in Escherichia coli in inclusion bodies that were solubilized and refolded to active protein. Binding of K1 MAb and FAb was compared by radioactive binding and competition assays and by surface plasmon resonance (BIAcore). Recombinant K1(FAb) binds to cells expressing K1-antigen with a similar affinity as papain derived FAb from K1(IgG) and with a 4- to 10-fold reduced affinity compared with bivalent IgG. The cloned FAb can be used to make higher affinity antibodies and immunoconjugates that could be useful for various types of immunotherapies.

Amino Acid Sequence↗
PubMed1997

Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation.

We have developed a novel method to stabilize a recombinant antibody Fv fragment. The V(H) and V(L) domains of this Fv fragment, called pFv (permutated Fv), are covalently interconnected to each other at the two "base-loops" that normally connect V(H) beta strand 3 to 3b and V(L) beta strand 3 to 3b. To produce the base-loop stabilized Fv fragment, we connected the N-terminal half of the V(L) domain (V(L) 1-40) of murine antibody anti-Tac to the C-terminal half of V(H) (V(H) 42-115). We also fused the C terminus of V(H) by a (Gly4Ser)3 linker to the N-terminal half of V(H) (V(H) 1-40, thereby generating a permutated V(H) domain). Finally we connected the base loop of V(H) (N-terminal half) to the C-terminal half of V(L) (V(H) 42-115). The anti-Tac pFv fragment was fused to a truncated form of Pseudomonas exotoxin to generate a pFv-immunotoxin. Fvs with the correct structure were produced by refolding of recombinant inclusion body protein using a renaturation protocol that was originally developed for Fab and scFv fragments. Due to the artificially connected and permutated primary sequence, the folding pathway for the pFv structure may possibly be different from the conventional folding of antibody domains. Analysis of antigen binding of anti-Tac pFv, and of the specific cytotoxicity of pFv-immunotoxin towards antigen expressing cancer cells demonstrated that the anti-Tac pFv retained most of its affinity and full specificity when compared to anti-Tac scFv. Also anti-Tac pFv was relatively stable, retaining 25% of its binding activity after a 24 hour incubation in human serum at 37 degrees C. This indicates that connection of base loops can be a useful alternative to linker or disulfide stabilization of Fv fragments.

Amino Acid Sequence↗
PubMed1997

Effects of BCL-2 overexpression on the sensitivity of MCF-7 breast cancer cells to ricin, diphtheria and Pseudomonas toxin and immunotoxins.

Immunotoxins are presently being evaluated as novel agents for cancer therapy. The direct mechanism by which immunotoxins kill cancer cells is inhibition of protein synthesis, but cytotoxicity due to induction of apoptosis has also been observed with these agents. Some cancers that express high levels of BCL-2 are relatively resistant to apoptosis inducing agents. It is therefore important to determine to what degree the toxicity of ricin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas exotoxin derived immunotoxins towards cancer cells can be attributed to inhibition of protein synthesis, and to what degree to subsequent induction of apoptosis. We compared the sensitivity of MCF-7 breast cancer cells that were stably transfected with a BCL-2 expression plasmid and thus protected against apoptosis and of MCF-7 cells transfected with a control plasmid towards ricin, diphtheria and Pseudomonas toxin, a Pseudomonas toxin-derived immunotoxin (LMB-7) and tumour necrosis factor alpha (TNF). We found that BCL-2 mediated inhibition of apoptosis renders the cells almost completely resistant (1000-fold) to tumour necrosis factor, but the same cells were only 3-10 fold more resistant to cytotoxicity induced by immunotoxin LMB-7 as well as Pseudomonas exotoxin, diphtheria toxin and ricin. We next studied several leukaemia cell lines with variable levels of BCL-2 expression and found them quite sensitive to a Pseudomonas exotoxin containing immunotoxin independent of the level of BCL-2. Our data indicate that although BCL-2 overexpression can have a modest effect on sensitivity to an immunotoxin, cell lines derived from patients are still very sensitive to immunotoxins.

Journal Article↗
PubMed1997

Localization of the cell proliferation and apoptosis-associated CAS protein in lymphoid neoplasms.

We have evaluated the expression and distribution of the cellular apoptosis susceptibility (CAS) protein in normal lymphoid tissue and malignant lymphomas. CAS protein, the product of the CAS gene, is associated with microtubules and the mitotic spindle. Immunohistochemistry with an antibody to CAS shows many CAS-positive cells in normal tonsils. The majority of strongly CAS-positive cells were localized to the dark zone of the follicles, whereas the mantle zone and interfollicular areas were essentially negative. Double staining for CAS and Ki-67 revealed co-expression of the two proliferation markers in approximately 85 to 90% of the CAS-positive cells. Different subtypes of lymphomas exhibited varying patterns of CAS expression. Low-grade non-Hodgkin's lymphoma generally revealed weak staining with CAS, with 10 to 60% of all cells being positive. In contrast, highly malignant non-Hodgkin's lymphoma and malignant cells of Hodgkin's disease displayed very strong CAS positivity, with staining of up to 80% of the atypical cells. Overall, the staining pattern of CAS and Ki-67 was superimposable within a particular lymphoma subtype. However, in all lymphomas we observed a significant fraction of CAS-positive normal and malignant lymphocytes that were Ki-67 negative, probably because they were momentarily noncycling cells. We conclude that a high expression of CAS correlates with proliferation of normal and malignant lymphoid cells. The fact that detection of CAS protein identifies a higher portion of proliferating and malignant cells than Ki-67 warrants further evaluation of CAS protein as a marker with a diagnostic potential.

Apoptosis↗