PubMed Health⌕ Search

Biomedical subjects

P E Petrides

Publications and source records attributed to P E Petrides.

At least 37 records · Page 2Linked to original sources
PubMed1997

Molecular analysis of Gaucher disease: distribution of eight mutations and the complete gene deletion in 27 patients from Germany.

Gaucher disease is the most common lysosomal storage disease with a high prevalence in the Ashkenazi Jewish population but it is also present in other populations. The presence of eight mutations (1226G, 1448C, IVS2+1. 84GG, 1504T, 1604T, 1342C and 1297T) and the complete deletion of the beta-glucocerebrosidase gene was investigated in 25 unrelated non-Jewish patients with Gaucher's disease in Germany. In the Jewish population, three of these mutations account for more than 90% of all mutated alleles. In addition, relatives of two patients were included in our study. Restriction fragment length polymorphism analysis and sequencing of PCR products obtained from DNA of peripheral blood leukocytes was performed for mutation analysis. Gene deletion was detected by comparison of radioactively labelled PCR fragments of both the functional beta-glucocerebrosidase gene and the pseudogene. Among the unrelated patients, 50 alleles were investigated and the mutations identified in 35 alleles (70%), whereas 15 alleles (30%) remained unidentified. The most prevalent mutation in our group of patients was the 1226G (370Asn-->Ser) mutation, accounting for 18 alleles (36%), followed by the 1448C (444Lcu-->Pro) mutation, that was found in 12 alleles (24%). A complete gene deletion was present in two alleles (4%). The IVS1+2 (splicing mutation), the 1504T (463Arg-->Cys) as well as the 1342C (409Asp-->His) mutations were each present in one allele (2%). None of the alleles carried the 84GG (frame-shift), 1604A (496Arg-->His) or the 1297T (394Val-->Leu) mutation. This distribution is different from the Ashkenazi Jewish population but is similar to other Caucasian groups like the Spanish and Portuguese populations. Our results confirm the variability of mutation patterns in Gaucher patients of different ethnic origin. All patients were divided into nine groups according to their genotype and their clinical status was related to the individual genotype. Genotype/phenotype characteristics of the 1226G, 1448C, and 1342C mutations of previous studies were confirmed by our results.

Adolescent↗
PubMed1996

HL-60 leukemia cells produce an autocatalytically truncated form of matrix metalloproteinase-9 with impaired sensitivity to inhibition by tissue inhibitors of metalloproteinases.

92-kDa type IV collagenase/gelatinase (matrix metalloproteinase-9; MMP-9; gelatinase B) expression and secretion has been shown to correlate with the invasive and metastatic potential of various malignant cells. MMP activity is tightly controlled by specific tissue inhibitors of metalloproteinases (TIMPs). We found the leukemic cell line HL-60 constitutively to release a 94-kDa gelatinase which we identified as MMP-9 shortened by nine amino acids at its N-terminal end. An additional gelatinolytic activity was present in small amounts and identified as a 63-kDa fragment of MMP-9 generated by autocatalytical processing. Both enzymes were identical regarding their N-terminus, indicating C-terminal truncation for the former. Incubation of cells with phorbol ester resulted in elevated amounts of both enzymes in conditioned media and in the secretion of TIMP-1. Both gelatinases were shown to be activated by trypsin and organomercurials and to possess similar activities towards various substrates. However, the 63-kDa enzyme differed from the 94-kDa enzyme in a significantly reduced inhibition by recombinant TIMP-1 and TIMP-2. Thus, the 63-kDa fragment of MMP-9 once activated may escape the regulatory influence of its specific inhibitors and may thereby promote matrix degradation during invasion of leukemic cells.

Amino Acid Sequence↗
PubMed1995

Insulin- and insulin-like growth-factor-I receptor tyrosine-kinase activities in human renal carcinoma.

We studied expression and functional characteristics of the insulin- and insulin-like-growth-factor-I (IGF-I) receptors in human renal carcinoma. Ligand-binding properties and tyrosine-kinase activity of both receptors, as well as the expression of the 2 isoforms of the human insulin receptor (HIR-A and -B) were analyzed in renal carcinoma and normal adjacent kidney tissue of 8 adult patients. Partially purified insulin- and IGF-I receptors from normal and renal cell carcinoma tissue possessed identical affinities for their ligands. Renal cell carcinoma, however, contained 3- to 4-fold more specific insulin-binding sites and 2-fold more IGF-I binding sites than adjacent normal kidney tissue. In addition, we determined the relative content of insulin/IGF-I receptor hybrids in both tissues. Renal cell carcinoma and adjacent normal tissue revealed similar amounts of insulin/IGF-I receptor hybrids, i.e., 44 +/- 8.2% of tracer IGF-I binding in normal tissue and 46 +/- 12.0% in renal cell carcinoma. When equal amounts of insulin- and IGF-I receptor protein were studied, we found significantly increased receptor autophosphorylation and elevated substrate phosphorylation in carcinoma tissue. To assess whether the differences in insulin-receptor tyrosine-kinase activity were caused by an altered pattern of insulin receptor isoform expression, we determined mRNA levels for HIR-A and -B. The 2 insulin receptor isoforms were, however, expressed in highly variable ratios in both normal and tumor tissue. Our experiments show that renal carcinoma expresses an elevated amount of insulin- and IGF-I receptor protein with increased specific autophosphorylation and tyrosine-kinase activity each. The increase of insulin-receptor tyrosine-kinase activity in renal carcinoma cannot be explained by an altered expression pattern of insulin receptor isoforms.

Carcinoma↗
PubMed1995

Cytokine regulation of matrix metalloproteinase activity and its regulatory dysfunction in disease.

Matrix metalloproteinases (MMPs) represent a family of structurally and functionally related enzymes responsible for the proteolytic degradation of extracellular matrix (ECM) components such as basement membrane or interstitial stroma. MMPs are important participants of normal tissue remodeling. Due to their potential hazardous effects MMPs are highly regulated at different levels. At the transcriptional level, MMP expression is precisely controlled by various cytokines acting through positive or negative regulatory elements of its genes. Moreover, MMP activity is post-transcriptionally regulated by proteolytic activation of the latent proenzymes and by interaction with specific tissue inhibitors of metalloproteinases (TIMPs). Expression and secretion of both MMP activating enzymes and TIMPs are also influenced by cytokines. Dysregulation of MMP production and activation may cause altered extracellular proteolysis that is associated with a number of diseases such as rheumatoid arthritis and tumor metastasis. Thus, the molecular analysis of the regulatory mechanisms of gene expression and activity of MMPs and their inhibitors is essential for understanding the complex scenario of tissue remodeling and ECM degradation under both normal and pathological conditions.

Animals↗
PubMed1995

Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs).

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.

Amino Acid Sequence↗
PubMed1994

Regulation of 92-kD gelatinase release in HL-60 leukemia cells: tumor necrosis factor-alpha as an autocrine stimulus for basal- and phorbol ester-induced secretion.

Matrix metalloproteinase 9 (MMP-9), also known as 92-kD type IV collagenase/gelatinase, is believed to play a critical role in tumor invasion and metastasis. Here, we report that MMP-9 was constitutively released from the human promyelocytic cell line HL-60 as determined by zymographic analysis. Tumor necrosis factor-alpha (TNF-alpha) enhanced the enzyme release threefold to fourfold and the protein kinase C (PKC) activator and differentiation inducer 12-O-tetradecanoylphorbol-13-acetate (TPA) eightfold to ninefold. Gelatinase induction by TNF-alpha and TPA was inhibited by actinomycin D or cycloheximide, indicating that de novo protein synthesis was required. Neutralizing monoclonal antibodies to TNF-alpha (anti-TNF-alpha) decreased the basal MMP-9 release of these cells. In addition, these antibodies also significantly interfered with the TPA-induced enzyme release. Agents that inhibit TNF-alpha expression in HL-60 cells, such as pentoxifylline and dexamethasone, completely abrogated both the constitutive and TPA-evoked MMP-9 release. Diethyldithiocarbamate, which is known to stimulate TNF-alpha production in HL-60 cells, exerted a positive effect on MMP-9 release in untreated cells but was inhibitory in TPA-treated HL-60 cells. The PKC inhibitor staurosporine at low concentrations (100 ng/mL) caused a significant augmentation of MMP-9 release in untreated cultures that was blocked by the addition of anti-TNF-alpha. High concentrations (2 mumol/L) of staurosporine completely abolished the extracellular enzyme activity both in untreated and TPA-stimulated cells. These results suggest, that TNF-alpha is required for basal and PKC-mediated MMP-9 release in HL-60 leukemia cells. Thus, MMP-9 secretion may be regulated by TNF-alpha not only in a paracrine but also in an autocrine fashion. This may potentiate the matrix degradative capacity of immature leukemic cells in the processes of bone marrow egress and the evasion of these cells into peripheral tissue.

Alkaloids↗
PubMed1994

Clinical importance of P-glycoprotein-related resistance in leukemia and myelodysplastic syndromes--first experience with their reversal.

P-glycoprotein (P-gp) expression in mononuclear bone marrow cells was analyzed in 119 patients, including 60 with chronic myelogenous leukemia (CML), 48 with myelodysplastic syndromes (MDS), and 11 with acute myelogenous leukemia (AML). For P-gp measurement an immunocytological method using monoclonal antibodies C219, 4E3, and MRK 16 and the reverse transcription-polymerase chain reaction technique were applied. According to our results obtained in healthy volunteers using the immunocytological method, the limit for P-gp overexpression was set at > or = 10% P-gp-positive mononuclear bone marrow cells and at > or = 30% P-gp-positive mononuclear peripheral blood cells. All 42 CML patients in chronic phase had normal P-gp expression. P-gp overexpression was demonstrated in four of six patients in accelerated myelogenous blast cell phase and in four of 12 CML-BC patients. Of eight CML patients in blast crisis (BC) with normal P-gp expression, partial remission was achieved in three and minor response in five after prednisone/vindesine therapy. All four of the 12 CML-BC patients with P-gp overexpression did not respond to this therapy. Normal P-gp expression was seen in 41 (85.4%) of 48 untreated MDS patients. While P-gp overexpression did not develop during therapy in any of the myelodysplastic syndrome patients treated with low-dose ara-C alone, four of eight treated with low-dose ara-C plus GM-CSF and four of 11 treated with low-dose ara-C and IL-3 developed P-gp overexpression after therapy. Furthermore, 11 AML patients at primary diagnosis, including five AML patients with P-gp overexpression, who were treated with idarubicin, vepesid, and cytarabine V (ara-C) showed a complete remission. Additionally, one daunorubicin-cytarabine-pretreated refractory AML patient was treated with the oral form of the P-gp modulator drug dexniguldipine and achieved complete remission for a duration of 7 months. Our results suggest that in CML patients in BC, P-gp expression influences outcome after therapy. Further more, studies in a larger series of patients are necessary to prove the efficacy and toxicity of idarubicin/vepesid and cytardbine--or dexniguldipine-containing--therapy in relation to P-gp expression of AML patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Cytogenetic and growth factor gene analysis of a renal carcinoma cell line.

Although there are many studies analyzing cytogenetic or molecular alterations of human renal primary tumors, there have only been a few reports addressing both questions on renal cell carcinoma (RCC) cell lines. We have therefore investigated an RCC cell line, namely KTCTL-26A, by banding techniques and simultaneous growth factor gene expression analysis. KTCTL-26A represents a well-defined stemline and sidelines in the near-diploid range with clonal aberrations involving chromosomes 2, 3, 5, 7, 9, 13, 16, 21, 22, and Y in structure and/or number. The predominant karyotypic changes were a partial loss of chromosome 3p (ie, 3p14) and a gain of copies of chromosome 7 (trisomy or partial tetrasomy). By Northern analysis, in KTCTL-26A we found underexpression of the proEGF-gene (located on chromosome 4) and overexpression of the genes for proTGF-alpha and the EGF-receptor, which are located on chromosomes 2 and 7, respectively. By Southern blot analyses there was no evidence for an amplification in the case of the EGF-R and proTGF-alpha genes. Because these changes of gene expression were observed in both the cell line and in primary kidney tumor samples, they seem to be of constitutive (and not adaptive) nature. Hence, KTCTL-26A can serve as a model for the study of the origin of these molecular alterations and as a preclinical model for their genetic manipulation (e.g., by using antisense-oligonucleotides) for therapeutic purposes.

Carcinoma, Renal Cell↗
PubMed1993

Detection of somatic changes in human renal cell carcinomas with oligonucleotide probes specific for simple repeat motifs.

The purpose of our study was to detect somatic changes in renal cell carcinoma by multilocus fingerprinting. DNA fingerprints were generated from the DNA of normal and malignant renal tissue samples of 29 patients with nonhereditary kidney carcinoma by using oligonucleotide probes specific for simple repeat motifs such as (GTG)5, (CA)8, (GACA)4, or (TTAGGG)3. Each probe rendered a typical fingerprint pattern, because it is specific with respect to the target regions recognized in the genome. The restriction enzymes used were HinfI and HaeIII. Changed banding patterns were detected by using (GTG)5 in 20% of the tumors, in 20% for (CA)8 after HinfI digestion, and in 10% after HaeIII digestion. Even more informative probes were (GACA)4, showing 70% changes after HaeIII digestion, and (TTAGGG)3, with 80% changes after digestion with either enzyme. Since the simple repeat motifs recognized by (GACA)4 are localized on the short arms of the acrocentric chromosomes (13, 14, 15, 21, and 22), it is possible that sequences important for renal carcinogenesis are present in these regions. The observation of changes in regions to which (TTAGGG)3 hybridizes points to an involvement of DNA elements in telomeric sequence related regions in human kidney tumor formation.

Adult↗
PubMed1992

Immunoreactive low-molecular-weight epidermal growth factor in urine of patients with renal cell carcinoma.

A specific heterogeneous enzyme-linked immunosorbent assay (ELISA) has been established in order to determine levels of low-molecular-weight epidermal growth factor (EGF) in the urine of patients with renal cell carcinoma who had undergone unilateral radical nephrectomy. Urine specimens, i.e., 20 pre- and postsurgical specimens from a group of patients and 22 from a control group, were assayed after the urine had been freed from high-molecular-weight proteins (greater than 30 kDa) and salts. EGF levels were expressed as urinary EGF/creatinine ratios, and a highly significant decrease (alpha = 0.0005 by Student's t-test) of urinary EGF was found in the patient group prior to surgery. The cancer patients also showed an additional loss of urinary EGF after unilateral nephrectomy (alpha = 0.0005 by Student's t-test). These data correlate with our previous findings that pro-EGF gene expression is decreased in human renal carcinoma and support the concept that low-molecular-weight urinary EGF is derived from high-molecular-weight kidney pro-EGF.

Biomarkers, Tumor↗