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R Kofler

Publications and source records attributed to R Kofler.

At least 55 records · Page 3Linked to original sources
PubMed1996

Glucocorticoid-receptor-gene defects and resistance to glucocorticoid-induced apoptosis in human leukemic cell lines.

The application of glucocorticoids (GC3) in human leukemia is based on apoptosis induction but is often hampered by GC resistance. To delineate resistance mechanisms, we examined 5 GC-resistant leukemic cell lines, termed CEM-C7.R1-R5, isolated from the GC-sensitive human acute-T-cell-leukemic line, CCRF-CEM-C7, by selection in GC-containing medium. GC resistance was ascertained by analyzing cell-cycle progression, proliferation, and apoptosis. Radioreceptor assays revealed absence of ligand-binding activity in all clones, suggesting that defects in GC-receptor(GR) expression cause GC resistance. Analyses of the GR gene revealed that all but one (CEM-C7.R5) of the clones were heterozygous for the previously described L753F mutation. CEM-C7.R5 was either hemi- or homozygous for the L753F mutation and, hence, lacked a functional GR. Sequencing of the allele not carrying the L753F mutation of the other GC-resistant sub-lines revealed additional mutations in the GR gene in 3 cases: CEM-C7.R1 and R2 had a base-pair deletion in exon 9 (deltaT740) that resulted in a reading-frame shift and a pre-terminal in-frame stop. Translation of this mutant mRNA would produce a protein lacking 32 amino acids and expressing 4 altered residues at its new C terminus. CEM-C7.R3 harbored a non-sense mutation (Q710X) in exon 8, and its mRNA would be translated into a protein lacking 67 residues. Only CEM-C7.R4 cells were devoid of mutations in the coding region of the L753F negative allele. These data suggest that, in the CCRF-CEM acute-lymphatic-leukemia model, mutations in the GR-gene coding region represent one, but not the only, cause of GC resistance.

Alleles↗
PubMed1996

Resistance to glucocorticoid-induced apoptosis in human T-cell acute lymphoblastic leukemia CEM-C1 cells is due to insufficient glucocorticoid receptor expression.

The ability of glucocorticoids (GCs) to induce death in lymphoid-origin cells is the basis for their frequent use in the therapy of various human hematological malignancies. However, the occurrence of primary or secondary GC resistance limits their clinical usefulness. Prior investigations into the mechanism of GC resistance in established human leukemic cell lines revealed loss-of-function mutations in the GC receptor (GR) gene. In this study, we analyzed the GC-resistant human acute T-cell leukemia line CEM-C1, which has been reported to express biochemically functional GR and, thus, was thought to owe its GC resistance to signal transduction changes distal from the GR. Radioligand binding assays revealed a 2-3-fold lower expression of GR in CEM-C1 than in the GC-sensitive sister cell line CEM-C7H2. Analysis of transcriptional activity using mouse mammary tumor virus-long terminal repeat-controlled chloramphenicol acetyltransferase expression in transient transfection assays confirmed the expression of functional GR in CEM-C1 but at levels lower than those in CEM-C7H2 cells. Upon molecular analyses of the GR gene and its transcripts, we found that CEM-C1 cells were heterozygous for the ligand binding domain L753F point mutation in exon 9, which is also present in GC-sensitive CEM-C7H2. No mutations, however, were found on the second GR allele of CEM-C1. To test the possibility that resistance in CEM-C1 cells might be caused by insufficient expression of GR, we established several cell lines stably transfected with rat GR expression vectors. These cell lines differed in exogenous GR expression as determined by Northern blotting and radioligand binding assays. The GR expression level in individual lines correlated well with their sensitivity to GC-induced apoptosis. Thus, GC resistance of CEM-C1 cells might be due to subthreshold expression of functional GR rather than defects in signal transduction pathways distal from the GR. Since several clinical investigations showed a correlation between reduced GR expression and poor response to GC-containing treatment, the CEM-C1 line may represent a valid model for GC resistance in human acute T-cell leukemia.

Animals↗
PubMed1996

The role of somatic mutation in the generation of the protective humoral immune response against vesicular stomatitis virus.

During most clinically relevant infections with cytopathic viruses, neutralizing antibodies are generated early, i.e., within the first week of infection. As early as 4 days after immunization of mice with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, hybridomas could be isolated that secreted virus-neutralizing IgGs. Such antibodies were devoid of somatic mutations, showed high binding avidities (approximately 10(9) M-1), and used V gene fragments predominantly belonging to the VHQ52 and VK19-28 families. In contrast, most secondary and hyperimmune response IgGs isolated 12 and 150 days after infection used several additional V gene combinations. These, which used the VHQ52/VK19-28 combination of early IgGs, were point mutated but showed only marginally enhanced binding avidities. Since all VHQ52/ VK19-28-positive IgGs bound to one subsite within the major antigenic site of VSV-G irrespective of the presence or absence of somatic point mutations, fine specificity diversification of secondary and hyperimmune responses was achieved by newly appearing V gene combinations.

Amino Acid Sequence↗
PubMed1996

Lack of association between atopy and the Ile181Leu variant of the beta-subunit of the high-affinity immunoglobulin E receptor.

A previous study has reported a strong association of a variant (Ile181Leu) of the beta-subunit of the high-affinity IgE receptor (Fc epsilon RI-beta) with allergic asthma bronchiale in a random patient sample. Based on their results the authors concluded that Fc epsilon RI-beta may be the maternally inherited, atopy-causing locus. We have investigated 40 unrelated atopic patients, 30 with allergic asthma and 10 with atopic dermatitis or allergic rhinoconjunctivitis along with some of their relatives for the presence of Ile181Leu by nucleic acid sequence analysis and/or hybridization with mutation-specific oligonucleotide probes. None of the probands showed this mutation suggesting that its association with atopy may be restricted to certain populations or occur at lower frequency than reported.

Adolescent↗
PubMed1996

CYP11B1 mutations causing congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.

Accurate knowledge of the molecular basis of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency is a prerequisite for genetic counseling, prenatal diagnosis, and treatment. Analysis of nine patients suffering from severe manifestations of this disorder led to the identification of seven novel mutations in their CYP11B1 genes. A Caucasian patient was homozygous for the missense mutation R448H, previously found only in Jews of Moroccan origin. An Iranian patient was found to be homozygous for a different mutation in the same codon, R448C. Of four unrelated patients, two were homozygous for a nonsense mutation (W247X), whereas two others were compound heterozygotes for W247X in combination with either R448H or E371G. Two other patients were homozygous for either the missense mutation A331V or an in-frame CTG insertion adjacent to codon 464 (InsCTG464). One patient was a compound heterozygote for two mutations in exon 2, a 28-bp deletion (delta 28bpEx2) and the missense mutation V129M. All of the missense mutations and the CTG insertion caused a complete loss of steroid 11 beta-hydroxylating activity when expressed in cultured cells. These data support previous suggestions of mutational hot spots in CYP11B1 and confirm that severe clinical manifestations are associated with complete loss of enzymatic activity.

Adrenal Hyperplasia, Congenital↗
PubMed1995

Overall and site-specific risk of malignant melanoma associated with nevus counts at different body sites: a multicenter case-control study of the German Central Malignant-Melanoma Registry.

A large number of benign melanocytic nevi is the major risk factor for malignant melanoma (MM). In a multicenter case-control study, the number of common (CN) and clinically atypical (AN) nevi were counted separately at individual sites in 278 melanoma patients and 278 age- and gender-matched non-melanoma controls. Relative risk (RR) adjusted for age and sex was calculated. In men as well as women, the number of CN on the legs was the best predictor of overall melanoma risk. In men, RR for developing MM when > or = 1 AN were present on the trunk was 4-fold (vs. none). In women, presence of AN on the arms increased RR 9.5-fold. For men and women combined, after adjusting for age and gender, the RR for developing MM on the trunk and on the legs was best predicted by counts of CN at the respective body region. However, high counts of CN on the arms were associated with high melanoma risk on the legs (somewhat lower on the trunk). For AN, no site-specificity of melanoma risk was found. Our data suggest that nevus counts of the legs are the best predictor of overall melanoma risk if total body nevus counts are not feasible. Although high counts of CN on the trunk and legs are associated with a higher risk of developing MM at the respective site than at another site, our data do not unequivocally support a direct site-specific melanoma risk.

Arm↗
PubMed1995

Splice site mutation in the glucocorticoid receptor gene causes resistance to glucocorticoid-induced apoptosis in a human acute leukemic cell line.

Induction of apoptosis is the molecular basis for the therapeutic application of glucocorticoids (GC) in human leukemia. The beneficial effect of endocrine therapy is, however, hampered by the occurrence of resistant clones evolving under selective GC pressure. To delineate molecular mechanisms of GC resistance, we PCR amplified, cloned, and sequenced GC receptor (GR) transcripts and gene segments from a GC-resistant subclone of the human acute leukemic cell line CCRF-CEM, termed CEM-R6. Our analyses revealed that one GR gene allele harbored a point mutation (L753F) previously shown to compromise GR functions in other CCRF-CEM derivatives. On the second allele, we identified an A to G point mutation in the 3'-splice junction of intron G. As a consequence, a cryptic splice site 8 base pairs downstream within exon 8 is recognized, which leads to an 8-base deletion in the GR mRNA, resulting in reading frame shift and 2 consecutive in-frame preterminal stop codons. Translation of this mutant mRNA would produce a truncated GR protein missing 93 amino acids of the ligand-binding domain and expressing 9 altered residues at its new COOH terminus. In concert with the L753F mutation on the other allele, this molecular defect explains the GC-resistant phenotype and provides further evidence for mutational GR gene inactivation as a mechanism for human leukemic cells to escape GC-induced apoptosis.

Amino Acid Sequence↗
PubMed1995

Clustered and interspersed gene families in the mouse immunoglobulin kappa locus.

Although numerous solitary germ-line V kappa genes and two small V kappa contiguously cloned gene regions (contigs) are known, no attempts to systematically elucidate the structure of the kappa locus of the mouse have been reported so far. As a first step to this aim we screened a cosmid library of C57BL/6J mouse DNA with 18 probes that are more or less specific for the different V kappa gene families. Ninety-one V kappa gene-containing cosmid clones were characterized by detailed restriction mapping and hybridizations. Several contigs were constructed from overlapping clones. The contigs and the still unlinked cosmid clones cover 1.6 Mb. Many of the cosmid clones were localized on chromosome 6 where the kappa locus is known to reside; no evidence for the existence of dispersed V kappa genes (orphons) was obtained. Eighty-five strong hybridization signals were assigned to distinct V kappa gene families, while for 11 weak signals the assignment was less definite. As to the distribution of gene families within the locus the following situation emerged: there are both, groups of genes which belong to one V kappa gene family ("clusters") and groups in which genes of different families are interspersed. The interspersion of gene families seems to be more pronounced than has been assumed so far. Additional V kappa genes which are known to exist will have to be isolated from other gene libraries of the same mouse Ig kappa haplotype.

Animals↗
PubMed1995

Computer simulations of histologic patterns in melanoma using a cellular automaton provide correlations with prognosis.

Computer simulations have been used frequently in the life sciences to investigate the mechanisms of morphologic pattern formation. The cellular automaton program SMN5 is designed to simulate tumor growth and to estimate biologic properties by comparing real tumor patterns with computer-simulated reference patterns. This method was applied to 195 cases of primary melanoma of the skin. S-100-stained sections were evaluated by image analysis and compared statistically to a reference set of 4000 simulated patterns. Estimates of tumor cell proliferation, motility, cell loss, cohesion, stroma destruction, and intercellular signals (autocrine and paracrine factors affecting growth, motility, and cell loss) were calculated. Twelve of 18 estimated parameters correlated significantly with tumor progression, as indicated by vertical tumor thickness (linear regression analysis: p < or = 0.05), and 13 of 18 parameters carried prognostic significance (log rank test: p < or = 0.05). Poor prognosis was associated particularly with a pronounced increase in the estimates of proliferation, tumor cell motility, and stromal degradation. Poor prognosis was also associated with a decrease in the estimates of cell loss, tumor cell cohesion, and paracrine growth factor dependence. In multivariate analysis using Cox's proportional hazard model, stromal degradation and motility showed prognostic information in addition to conventional prognostic parameters. The study shows that analytical comparison of real tumors with computer-simulated patterns of a cellular automaton facilitates a functional interpretation of tumor morphology, which carries prognostic significance in cutaneous melanoma.

Computer Simulation↗
PubMed1995

Glucocorticoid-mediated immunomodulation: hydrocortisone enhances immunosuppressive endogenous retroviral protein (p15E) expression in mouse immune cells.

To define glucocorticoid (GC)-regulated genes contributing to the anti-inflammatory and immunosuppressive effects of GC, previous work from our laboratory revealed up-regulation of transcripts from endogenous type B mouse mammary tumour virus (Mtv) and type C murine leukaemia virus (Emv) loci by high dose GC treatment of P388D1 macrophage-like cells. This study demonstrates enhancement of expression from Mtv and Emv loci in P388D1 cells by more physiological hydrocortisone concentrations (1 microM), and shows direct transcriptional mode of regulation by blocking GC-mediated signal transduction at different levels. Furthermore, we found up-regulation of Emv mRNA steady-state levels in murine lymphoid lineage cells (T-like EL4 and BW5147 cells; B-like X63 cells) upon GC treatment. The Emv transcripts shown by us to be GC-up-regulated encode for the transmembrane envelope protein TM/p15E which is highly conserved in several retroviruses. TM/p15E and the p15E-like products found in humans exert immunosuppressive effects in different test systems. Thus, our findings raise the possibility that immunomodulation by GC might be mediated in part by enhanced expression of p15E(-like) products.

Adjuvants, Immunologic↗
PubMed1995

Amino acid substitution R384P in aldosterone synthase causes corticosterone methyloxidase type I deficiency.

Corticosterone methyloxidase type I (CMO-I) deficiency is an autosomal recessively inherited disorder causing congenital hypoaldosteronism due to defects in aldosterone synthase (P450aldo), the enzyme that converts 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone, and aldosterone. To clarify the molecular basis of CMO-I deficiency and gain further insight into the structure-function relationship of P450aldo, we cloned and sequenced the CYP11B2 gene (encoding P450aldo) of a male Caucasian patient suffering from CMO-I deficiency and identified a single point mutation leading to substitution of the highly conserved arginine-384 by proline (R384P). Differential hybridization of mutation-specific oligonucleotide probes to polymerase chain reaction-amplified CYP11B2 fragments revealed that both parents were heterozygous carriers for R384P, whereas the patient appeared homozygous. The patient's healthy brother and 85 individuals without known aldosterone synthase deficiency did not carry the R384P mutation. Introduction of this mutation into a CYP11B2 complementary DNA expression vector construct and subsequent expression in COS cells revealed that R384P leads to complete loss of 11 beta- and 18-hydroxylase activities of P450aldo. Thus, the R384P mutation provides a molecular explanation for the CMO-I deficiency in this patient and suggests that arginine-384 plays a major role in P450aldo function.

Amino Acid Sequence↗
PubMed1994

[Late metastasis of malignant cutaneous melanoma].

Late recurrence of malignant melanoma, defined as the occurrence of metastases after a disease-free interval of 10 or more years, is a rare but well-known clinical phenomenon. We report on three female patients who developed in transit metastases 26, 15 and 10 years after excision of the primary tumour. All three patients are disease-free 8 and 9 years after resection of the metastases. Reports in the literature also indicate a favourable prognosis for patients with local recurrence or in transit or regional lymph node metastases. The prognosis for patients with late distant metastases, however, is generally poor. For the first group, an early therapeutic intervention is indicated. This and the occurrence of second and multiple primary melanomas make lifelong aftercare desirable. On the other hand, long-term aftercare of the constantly increasing number of melanoma patients will soon exceed the capacity of existing facilities. A high-risk group for late recurrence that would profit by long-term aftercare cannot be defined at the moment.

Adult↗
PubMed1994

Melanotic macules following Blaschko's lines in McCune-Albright syndrome.

Hyperpigmented macules are a characteristic feature of neurofibromatosis and the McCune-Albright syndrome. Whereas neurofibromatosis 1 has an autosomal dominant mode of inheritance, it has been suggested that McCune-Albright syndrome is the result of a lethal gene surviving by mosaicism. Recent molecular studies have supported this concept by providing evidence of a somatic mutation of the gene encoding the G protein. We report two patients with McCune-Albright syndrome whose melanotic macules show a clear relation to the lines of Blaschko. The lines of Blaschko are thought to represent the dorso-ventral outgrowth of two different cell populations during embryogenesis, thus reflecting genetic mosaicism. A survey of published photographs of patients with McCune-Albright syndrome in the literature revealed additional cases with macules following Blaschko's lines. In other cases, the configuration of the macules was reminiscent of the flag-like rectangular pattern of pigmentation found in human chimaeras. A very early somatic mutation may have similar effects on the pigmentation pattern as a chimaeric state, which is the result of the double fertilization of an ovum. Café-au-lait spots in 10 of our own patients with neurofibromatosis 1 could not be associated with either Blaschko's lines or the rectangular pattern of pigmentation in chimaeras. We conclude that, in contrast with the café-au-lait spots in autosomal dominant neurofibromatosis 1, the configuration pattern of melanotic macules in McCune-Albright syndrome in many cases characteristically reflects the mosaic state of the organism.

Child, Preschool↗
PubMed1993

Comparison of proliferative activity as assessed by proliferating cell nuclear antigen (PCNA) and Ki-67 monoclonal antibodies in melanocytic skin lesions. A quantitative immunohistochemical study.

Immunostaining with the monoclonal antibodies PCNA and Ki-67 provides a simple method for the assessment of growth fractions of tumors. Contrary to Ki-67, PCNA antibody can be applied on aldehyde- or alcohol-fixed and paraffin-embedded tissues, thus allowing studies on archival material. For 77 melanocytic skin lesions, we compared PCNA immunostaining on formalin-fixed tissue with Ki-67 immunostaining on frozen material of the same lesion. 16 benign melanocytic nevi (BMN, from 16 patients), 43 primary malignant melanomas (PMM, 42 patients), and 18 skin metastases of malignant melanoma (MMM, 12 patients) were included in the study. Maximum nuclear density (NDmax) of PCNA- and Ki-67-positive nuclei was assessed using interactive image analysis. NDmax values for both PCNA and Ki-67 differed significantly between the three diagnostic groups (Kruskal-Wallis H-test: p << 0.001). Mean values (given as 1000 nuclei/mm3 tissue) increased considerably from benign to malignant lesions (PCNA: BMN: 23.8 +/- 28.4 [mean +/- standard deviation], PMM: 48.1 +/- 41.0, MMM: 117.0 +/- 64.6; Ki-67: BMN: 6.4 +/- 3.3, PMM 25.0 +/- 31.1, MMM: 95.2 +/- 47.2). Correlation between NDmax values of PCNA- and Ki-67-positive nuclei was significant (Linear regression analysis: r = 0.51, p << 0.001). Furthermore, for PMM a significant correlation between histologic parameters related to prognosis (Breslow index and mitotic rate) and PCNA as well as Ki-67 expression was found (PCNA-Breslow index: r = 0.42, p < 0.01; Ki-67-Breslow index: r = 0.60, p << 0.001; PCNA-mitotic rate: r = 0.40, p < 0.01; Ki-67-mitotic rate: r = 0.50, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Tumor cell proliferation and motility estimates are prognostic factors in malignant melanoma.

Metastatic spread of solid tumors depends on a complex metastatic cascade. Metastatic tumor cells must therefore express a variety of biological properties including the ability to proliferate and the ability to migrate. In the present study, the prognostic significance of both parameters was investigated in 184 cases of malignant melanoma of the skin. Proliferative activity was assessed by mitotic counts, the degree of cell motility was estimated by comparing the histological pattern of the tumor with patterns obtained by computer simulations with predefined values of cell motility. Follow up of surviving patients was at least 5 years. Statistical analysis was done using classification and regression tree (CART) analysis combining mitotic counts and motility estimates. As far as overall survival was concerned, mitotic counts were the most significant variable separating one group of patients with mitotic counts < 17/mm2 and a 5-year survival rate of 82% and a group with mitotic counts > or = 17/mm2 and a 5-year survival rate of 52%. Within the group with low mitotic counts, motility estimates found a subgroup of patients with a coefficient of motility > or = 126, which had a 5-year survival rate of 55% similar to those patients with high mitotic counts. Corresponding results were obtained in the assessment of metastasis-free interval. The data show that not only proliferation as assessed by mitotic counts, but also estimates of tumor cell motility based on image analysis and computer simulations provide prognostic information in primary malignant melanoma of the skin.

Cell Division↗
PubMed1992

Mechanism of allergic cross-reactions--III. cDNA cloning and variable-region sequence analysis of two IgE antibodies specific for trinitrophenyl.

As a first step toward defining the molecular interactions between ligands and the IgE antigen-combining site, we report here the cDNA cloning and variable (V) region nucleic acid sequences of the heavy (H) and light (L) chains of 2 monoclonal mouse IgE antibodies to trinitrophenyl (ATCC-TIB142 = IGELa2 and ATCC-TIB141 = IGELb4). In all instances, full-length cDNA clones were obtained to facilitate future expression studies. The H chains were encoded by VH genes from the VH3660 and J558 gene families in context with DQ52 and DSP2.2 diversity (D) mini genes, and JH3 and JH4 joining (J) gene segments, respectively. Vk8/Jk2 and Vk1/Jk5 rearrangements encoded the respective L chain V-regions. Both antibodies exhibited considerable conservation of complementarity determining region (CDR) sequences, which will facilitate template-based computer modeling of the three-dimensional structures of complexes formed between various ligands and these antibodies. From sequence comparison between the dinitrophenyl (DNP)-binding myeloma protein MOPC-315 and these IgE antibodies likely candidates for hapten-contact residues within the binding sites of IGELa2 and IGELb4 have been suggested.

Amino Acid Sequence↗