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Biomedical subjects

P Hilgard

Publications and source records attributed to P Hilgard.

At least 19 recordsLinked to original sources
PubMed2000

A novel casein kinase 2 alpha-subunit regulates membrane protein traffic in the human hepatoma cell line HuH-7.

A previously isolated endocytic trafficking mutant (TRF1) isolated from HuH-7 cells is defective in the distribution of subpopulations of cell-surface receptors for asialoorosomucoid (asialoglycoprotein receptor (ASGR)), transferrin, and mannose-terminating glycoproteins. The pleiotropic phenotype of TRF1 also includes an increased sensitivity to Pseudomonas toxin and deficient assembly and function of gap junctions. HuH-7xTRF1 hybrids exhibited a normal subcellular distribution of ASGR, consistent with the TRF1 mutation being recessive. A cDNA expression library derived from HuH-7 mRNA was transfected into TRF1 cells, which were subsequently selected for resistance to Pseudomonas toxin. Sequence analysis of a recovered cDNA revealed a unique isoform of casein kinase 2 (CK2), CK2alpha". Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2alpha expression. Consistent with this finding, the hybrids HuH-7xHuH-7 and HuH-7xTRF1 expressed equivalent amounts of total CK2alpha. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2alpha and CK2alpha' and the novel CK2alpha" isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2alpha and CK2alpha', the mutant did not express CK2alpha". Based on the genomic DNA sequence, RNA transcripts encoding CK2alpha" apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2alpha or the newly cloned CK2alpha" restored the parental HuH-7 phenotype, including Pseudomonas toxin resistance, cell-surface ASGR binding activity, phosphorylation, and the assembly of gap junctions. This study suggests that HuH-7 cells express at least three CK2alpha isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.

Amino Acid Sequence↗
PubMed2000

Heparan sulfate proteoglycans initiate dengue virus infection of hepatocytes.

Dengue viruses (DEN) cause a broad spectrum of clinical manifestations including potentially life-threatening conditions such as hemorrhagic shock syndrome and less frequently acute hepatitis with liver failure and encephalopathy. In addition, dengue viruses provide a potential model to understand the initiation of hepatocyte infection by the structurally closely related hepatitis C virus (HCV), because this virus at present cannot be grown in cell culture. Although the initial steps of viral infection are a critical determinant of tissue tropism and therefore pathogenesis, little is known about the molecular basis of binding and endocytic trafficking of DEN or of any other flavivirus. Our studies revealed that binding of radiolabeled DEN to the human hepatoma cell line HuH-7 was strictly pH dependent and substantially inhibitable by the glycosaminoglycan heparin. Ligand-blot analysis, performed as a viral overlay assay, showed two heparan sulfate (HS) containing cell-surface binding proteins resolving at 33 and 37 kd. Based on the sensitivity of unprotected virus and the viral binding site on the cell surface to trypsin, viral internalization was quantified as an increase in trypsin protected virus over time. Virus trafficking to the site of degradation was inhibited by pH dissociation of the clathrin coat and dependent on IP(3)-mediated homotypic endosomal fusion. These findings confirm the hypothesis that binding and internalization of DEN by hepatocytes are mediated primarily by HS containing proteoglycans and suggest that flaviviruses traffic the major clathrin-dependent endocytic pathway during infection.

Carrier Proteins↗
PubMed1998

Trial of oral miltefosine for visceral leishmaniasis.

BACKGROUND: There is no effective oral treatment for visceral leishmaniasis (kala-azar), a disseminated intracellular protozoal infection that occurs worldwide. Miltefosine, an alkyl phospholipid developed as an oral antineoplastic agent, is active against visceral infection in animal models. We tested safety, tolerance, and efficacy of miltefosine in kala-azar. METHODS: Oral doses of miltefosine were given to six groups of five Indian men for 28 days: 50 mg every second day (group 1), 100 mg every second day (group 2), 100 mg/day (group 3), 150 mg/day (group 4), 200 mg/day (group 5), and 250 mg/day (group 6). Assessment for apparent cure--taken as an afebrile state with decreased spleen size and a splenic-aspirate parasite-density score of 0--was done on days 14 and 28. Definitive cure at 8 months required a parasite-free bone-marrow aspirate and no clinical evidence of relapse. FINDINGS: 21 of 30 patients were apparently cured on day 14. Transient episodes of vomiting and diarrhoea, were common during weeks 1-2 and were seen in 22 patients. Four other patients in groups 5 and 6 had miltefosine withdrawn after 7-10 days because of vomiting. One patient in group 6 developed renal insufficiency and severe diarrhoea and died on day 21. On day 28, all 29 remaining patients were apparently cured. By 8 months, seven of ten patients in groups 1 and 2 had relapsed; however, 18 of 19 patients treated daily (groups 3-6) appeared to be cured. Among the 21 definitive cures were the four patients treated for 10 days or less and 12 for whom previous therapy with pentavalent antimony had failed. INTERPRETATION: Treatment with miltefosine at 100-150 mg/day for 4 weeks has promise as an effective oral treatment of visceral leishmaniasis including antimony-resistant infection.

Administration, Oral↗
PubMed1998

[The "nutcracker syndrome" of the renal vein (superior mesenteric artery syndrome) as the cause of gastrointestinal complaints].

HISTORY AND CLINICAL FINDINGS: Since the age of 19 a now 22-year-old man had complained of intermittent abdominal pain, irregular stools and paroxysmal tachycardia. The only preceding illness had been a single episode of iron-deficiency anemia. A laparoscopy, done 8 months after the onset of symptoms, had revealed an inflamed Meckel's diverticulum which was surgically removed. After transient improvement the symptoms recurred 5 months postoperatively. On admission to clarify the cause of the symptoms he had discrete abdominal pain on pressure, but physical examination was otherwise unremarkable. INVESTIGATIONS: Routine biochemical tests and endoscopy were normal. Abdominal computed tomography was suspicious of severe narrowing of the left renal artery by a crossing superior mesenteric artery. As a result the left testicular vein and the peripelvic venous network were markedly dilated by retrograde congestion, strongly suggesting the "nutcracker syndrome" of obstruction of the left renal vein. This diagnosis was confirmed by selective renal phlebography and pressure measurement. TREATMENT AND COURSE: The vascular anomaly was corrected surgically by reimplanting the left renal vein into the inferior vena cava 3-4 cm further caudally. The patients has been completely symptom-free since then. CONCLUSIONS: The nutcracker-syndrome is a rare cause of hematuria. The coexistence of this anomaly with gastrointestinal symptoms has not been previously described, but it is likely that congestion of the splanchnic veins by obstruction of the left renal vein was at least partly responsible for them, in view of the postoperative relief.

Abdominal Pain↗
PubMed1998

Gene trapping identifies inhibitors of oncogenic transformation. The tissue inhibitor of metalloproteinases-3 (TIMP3) and collagen type I alpha2 (COL1A2) are epidermal growth factor-regulated growth repressors.

A gene trap strategy has been used to identify genes that are repressed in cells transformed by an activated epidermal growth factor (EGF)/EGF receptor signal transduction pathway. EGF receptor-expressing NIH3T3 cells (HER1 cells) were infected with a retrovirus containing coding sequences for the human CD2 antigen and for secreted alkaline phosphatase in the U3 region. By selecting for and against CD2 expression, we obtained clones in which the gene trap had integrated into genes selectively repressed by EGF. Two of these clones encoded for the secreted extracellular matrix proteins TIMP3 and COL1A2. We show here that both genes are downstream targets of RAS and are specifically repressed by EGF-induced transformation. Moreover, this strategy tags tumor suppressor genes in their normal chromosomal location, thereby improving target-specific screens for antineoplastic drugs.

3T3 Cells↗
PubMed1997

Characterization of gonadotropin-releasing hormone analogs based on a sensitive cellular luciferase reporter gene assay.

A novel cellular assay for the functional characterization of agonistic and antagonistic analogs of gonadotropin-releasing hormone (GnRH) was developed. This assay is based on a fusion of the c-fos immediate-early gene promoter to Photinus pyralis luciferase (Luc) as a reporter gene, stably transfected in a recombinant cell line expressing the human GnRH receptor. Transcription of endogenous c-fos and fos-Luc fusion gene are transiently induced quite similar by fetal calf serum or the superagonistic analog [D-Trp6] GnRH in a selected cell line. The reporter gene was therefore used to monitor agonist-induced signaling via the human GnRH receptor. Whereas Luc activity was induced in a dose-dependent manner by GnRH or [D-Trp6] GnRH, different antagonistic peptides completely inhibited this stimulation. The antagonistic potency (IC50) of various peptides with Cetrorelix and Antarelix as lead compounds in general correlated well with the binding affinity (KD) as determined from ligand binding experiments. The specificity of an inhibitory effect was confirmed by GnRH receptor-independent stimulation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate or basic fibroblast growth factor. Since this new reporter gene assay is sensitive and simple and can be performed in a microtiter plate, it will significantly facilitate screening and functional characterization of GnRH analogs.

Animals↗
PubMed1997

The development of alkylphosphocholines as signal transduction inhibitors: experimental and clinical challenges.

Alkylphosphocholines are a new class of anticancer agents. Their mode of action is considered to be related to the inhibition of phospholipase C and protein kinase C. These enzymes play a major role in intracellular signalling pathways. Their inhibition by alkylphosphocholines leads in the dimethylbenzanthracene-induced mammary carcinoma of the rat to a response pattern similar to that of the antiestrogen zindoxifene. This suggests that the inhibition of transcription factor formation might be the common pathway for alkylphosphocholines and antihormones. Based on the experimental dose-response pattern, new clinical strategies for dose finding and response evaluation will have to be developed for inhibitors of signal transduction, such as alkylphosphocholines.

Animals↗
PubMed1997

D-21266, a new heterocyclic alkylphospholipid with antitumour activity.

The aim of this study was to determine the antitumour effects of D-21266 in a rodent tumour model. Hexadecylphosphocholine (INN: Miltefosine) represents the first anticancer agent which was specifically formulated for topical use in cancer patients. The development as an oral drug was hampered by the gastrointestinal toxicity. Hexadecylphosphocholine derivatives were sought with a better therapeutic index. Octadecyl-(1,1-dimethyl-4-piperidylio) phosphate (D-21266) was identified as a suitable candidate. This compound is highly active in vitro inhibiting the growth of a number of human cancer cell lines. Mammary carcinomas were induced in Sprague-Dawley rats using DMBA, and oral doses of D-21266, in various schedules, were given to the animals. A high antineoplastic potency was observed without inducing loss of body weight at highly effective doses. The antitumour effect could be enhanced by introducing a dose schedule consisting of a high loading dose followed by a low maintenance dose, both of which are only marginally active when given alone. Therefore, D-21266 with its favourable pharmacological and toxicological profile, warrants evaluation in the clinic. However, the concept of clinical trials requires new approaches to dose finding and response evaluation, because the dose-response relationship of this compound is distinctly different from that of classical cytostatic agents.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Reversible tumorigenesis in mice by conditional expression of the HER2/c-erbB2 receptor tyrosine kinase.

In the present study we describe the reversible transformation of NIH3T3 fibroblasts by overexpression of the HER2/c-erbB2 receptor tyrosine kinase. Cell lines expressing HER2 under control of a tetracycline-responsive promoter were isolated. Induction of HER2 expression resulted in cellular transformation in vitro as depicted by growth in soft agar and focus formation in tissue culture. Subsequent treatment of these cells with the effector anhydrotetracyline switched-off HER2 expression and induced morphological and functional changes characteristic for non-transformed cells. Subcutaneous transplantation of cells in nude mice resulted in the formation of solid tumors. Interestingly tumor formation was completely suppressed by treatment of the animals with anhydrotetracyline. Our findings indicate that overexpression of HER2 induces the transformed phenotype of NIH3T3 cells and is required for tumor formation and progression in nude mice. By linking the expression of the marker gene secreted placental alkaline phosphatase to the expression of HER2, a sensitive monitoring of tumor development in nude mice was feasible.

3T3 Cells↗
PubMed1996

Pharmacological studies with cetrorelix (SB-75), a potent antagonist of luteinising hormone-releasing hormone.

The antitumour and hormone-suppressive effects of the luteinising hormone-releasing hormone LH-RH antagonist Cetrorelix (D-20761) and its pamoate salt (D-20762) were investigated in the model of the DMBA-induced mammary carcinoma of female rats and by testosterone determinations in normal male rats. Treatment with single high doses of D-20761 induced a rapid decrease of tumour weights with a dose-dependent duration of action. Strong antitumour effects were also observed by applying different multiple dose schedules, including a initial high dose (3.16 mg/kg, s.c.) followed by a low maintenance dose (31.6 micrograms/kg, s.c.). The stability of the molecule against degrading enzymes led to the idea of using the poorly soluble pamoate salt for facilitating a sustained release of active compound. This salt indeed induced a prolonged suppression of tumour growth and of testosterone levels. In conclusion, we found that Cetrorelix is a highly effective LH-RH antagonist which should be further developed for the treatment of hormone-dependent diseases.

Animals↗
PubMed1995

Selection and characterization of mammalian cell lines with stable over-expression of human pituitary receptors for gonadoliberin.

The cDNA encoding the receptor for gonadoliberin (GnRH or LH-RH) was isolated from a human pituitary cDNA library and heterologously expressed in the murine fibroblast cell line LTK-. By using a dicistronic expression strategy utilizing the internal ribosomal-entry-site sequence of poliovirus, single cell clones with stable and high expression of human gonadoliberin receptors were selected. In radioligand saturation-binding experiments, the gonadoliberin antagonist Cetrorelix showed high-affinity binding to the heterologously expressed human gonadoliberin receptor with a Kd of 0.1 nM. The pharmacological profile using 125I-Cetrorelix as radioligand and the authentic gonadoliberin or agonistic and antagonistic derivatives as competitors, showed a distinct rank order of binding potencies. Superagonistic gonadoliberin derivatives had more than ten-times higher binding affinities in comparison to gonadoliberin with a Kd of 3.47 nM. The gonadoliberin receptor expressed in stably transfected LTK- cells coupled to the inositol phosphate signal-transduction pathway. Gonadoliberin stimulated the synthesis of inositol 1,4,5-trisphosphate in a dose-dependent way with an EC50 of 5 nM. This stimulatory effect of gonadoliberin was completely antagonized by Cetrorelix in equimolar concentrations, demonstrating the high potency of this competitive receptor antagonist. In growth-arrested cells, a transient expression of the c-fos protooncogene was induced by gonadoliberin or [D-Trp6]gonadoliberin, showing that the gonadoliberin receptor couples to a putative mitogenic signal-transduction pathway in this heterologous cell system.

Amino Acid Sequence↗
PubMed1995

Induction of the c-myc but not the cH-ras promoter by platinum compounds.

The recombinant plasmids p324, p330 and p323 carrying the aminoglycoside phosphotransferase (aph) gene and 5' flanking c-myc sequences linked to the reporter gene chloramphenicol acetyl-transferase (cat) were introduced into the mouse erythroleukaemic cell line F412B2TK- and stable transfectants resistant to geneticin G418 were obtained. The effects of cis-platin and two novel platinum compounds, D19466 (lobaplatin) and D17872, on c-myc promoter regions were studied using a large range of drug concentrations and correlated with cytotoxicity data. It was found that cis-platin and D19466 show a similar pattern of cytotoxicity and activation of c-myc promoter-driven cat gene expression with maximum effect (increased expression of 7.4- and 8.1-fold, respectively) at a concentration of 5 x 10(-5) M, while the less cytotoxic D17872 only slightly activates cat expression at the same concentration. However, when the F412B2TK- cell line was transfected with a plasmid carrying 5' flanking sequences of the c-Hras1 gene with promoter/enhancer function, linked to the cat reporter, no similar inductive effect was observed with any of the platinum drugs used. These data suggest that platinum compounds and possibly other DNA-damaging agents may specifically influence the expression of certain genes.

Animals↗
PubMed1995

D-19575--a sugar-linked isophosphoramide mustard derivative exploiting transmembrane glucose transport.

D-19575 is a glucose derivative of ifosfamide mustard with a broad spectrum of antitumor activity in animal models. In comparison with ifosfamide, D-19575 is less toxic and is better tolerated by tumor-bearing animals, achieving a better therapeutic efficacy. D-19575 is directly cytotoxic in vitro--in contrast to ifosfamide--and it is possible to modulate this cytotoxicity by inhibition of transmembrane glucose transporters. Correspondingly, renal reabsorption of filtered D-19575 could be blocked by pre- and cotreatment with phlorizin, resulting in a higher urinary excretion of the unchanged drug. The toxicity to white blood cells, colony-forming units (CFU-C), and spleen-cell colony-forming units (CFU-S) is considerably lower for D-19575 as compared with ifosfamide. In conclusion, D-19575 is a new alkylating cytotoxic agent with increased antitumor selectivity, probably caused by an active transmembrane transport mechanism.

Absorption↗
PubMed1995

Opposite effect of miltefosine on the antineoplastic activity and haematological toxicity of cyclophosphamide.

The effect of pretreatment with miltefosine (MIL) on the antineoplastic activity of cyclophosphamide (CPA) was evaluated in subcutaneous benzo(a)pyrene-induced sarcomas (BPS) of the rat. MIL alone had no antineoplastic effect on this autochthonous tumour, but enhanced the chemotherapeutic effect of CPA. Conversely, MIL counteracted the myelotoxicity of CPA in normal adult rats. Although the nadir of the leucocyte count remained unchanged, the recovery phase was considerably shortened, an effect which resembled the pharmacological action of GM-CSF.

Animals↗
PubMed1995

Synthesis and antitumor activity of two ifosfamide analogs with a five-membered ring.

Two ifosfamide (CAS 3778-73-2) analogs with a five-membered ring, i.e. the oxazaphospholidine derivatives 6 and 7, were synthesized and their cytotoxic activity in vitro, acute toxicity and antitumor activity in vivo determined in comparison with the oxazaphorinane ifosfamide 1. The observed low biological activity gives evidence that both, the six-membered oxazaphosphorinane ring and the two N-2-chloroethyl-side chains are necessary for the generation of the ultimate alkylator, i.e. the ifosfamide mustard 5.

Animals↗
PubMed1994

Molecular and cellular effects of hexadecylphosphocholine (Miltefosine) in human myeloid leukaemic cell lines.

The molecular and cellular effects of the anti-neoplastic alkylphospholipid hexadecylphosphocholine (Miltefosine, MIL) on parameters associated with growth and differentiation of human myeloid leukaemic cell lines U937, KG1 and KG1a were investigated. On a cellular level, MIL has dose-dependent differentiation-inducing growth-promoting and cytotoxic activities exemplified by induction of respiratory burst activity, stimulation of interleukin-3 (IL-3)/granulocyte-macrophage colony stimulating factor (GM-CSF)-dependent growth of the KG1 cell line in soft agar culture, inhibition of cellular net growth and finally cell death. By northern blot analysis, transcription of functional receptors for IL-3, GM-CSF, G-CSF and FcRI were studied. It was shown that MIL has stimulatory activity on IL-3 and GM-CSF receptor gene transcription. In addition, the transcription of proliferation- and differentiation-associated proteins, namely histone subtypes, c-myc and NF-kappa B p50, were studied. MIL suppressed c-myc and enhanced NF-kappa B p50 transcription in the U937 cell line, comparable to the well-characterised differentiation-inducing phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA). We conclude that the interaction of MIL with its molecular target(s) in myeloid cells induces molecular and cellular effects associated with induction of differentiation, distinct from its cytotoxic activity.

Antineoplastic Agents↗