Biomedical subjects

L M Neckers

Publications and source records attributed to L M Neckers.

At least 37 records · Page 2Linked to original sources

PubMed1997

The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.

Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the gamma-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1-221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.

Adenosine Diphosphate↗

PubMed1997

Expression of PAX3 in Ewing's sarcoma family of tumors.

The Ewing's sarcoma family of tumors (ESFT) is the second most common pediatric malignancy originating in the bone and is characterized by the t(11; 22) translocation. PAX3, a member of the paired box family of genes, is expressed during embryonal development of neural crest cells and is involved in the t(2; 13) translocation found in alveolar rhabdomyosarcoma. Since ESFTs are believed to be derived from neural crest tissue, we screened a series of Ewing's sarcoma and peripheral neuroectodermal tumor cell lines and tumor specimens for expression of PAX3. We found expression of PAX3 in most, but not all, of the specimens analyzed, including cell lines and patient material.

Animals↗

PubMed1996

Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway.

c-Raf-1 (Raf-1) is a central component of signal transduction pathways stimulated by various growth factors, protein kinase C, and other protein kinases. Raf-1 activation is thought to be initiated at the plasma membrane after its recruitment by Ras. Raf-1 activation is associated primarily with proliferation and cell survival, but it has also been implicated in apoptosis. Raf-1 has also been shown to form complexes with both R-Ras and Bcl-2, raising the possibility that this component of cellular Raf-1 plays a role in apoptosis. Recently, taxol was reported to induce Bcl-2 phosphorylation and inactivation. We have previously demonstrated Raf-1 activation following taxol in MCF7 cells. We now present evidence that taxol fails to stimulate either apoptosis or phosphorylation of Bel-2 in the absence of Raf-1. Moreover, Raf-1 activation by taxol coincided with Bel-2 phosphorylation, showing similar dose and time dependence. Thus, our data support a role for a distinct subcellular component of Raf-1, which is taxol but not phorbol myristate acetate sensitive, in mediating an apoptotic pathway involving Bc1-2.

Antineoplastic Agents, Phytogenic↗

PubMed1996

bcl-2 protein downregulation is not required for differentiation of multidrug resistant HL60 leukemia cells.

Parental and multidrug resistant HL60 leukemia cell lines were used to study coupling of expression of apoptotic/cytostatic (bcl-2, bax, bclxL, p21/Waf1, and c-myc) genes during differentiation. The multidrug resistant HL60 cell line, HL60/ADR, was less sensitive than parental cells to cytostatic activity of low (0.4-2 ng/ml) doses of PMA. However, during treatment with standard differentiating doses of PMA (10 ng/ml), no difference between the two cell lines in cytostasis and differentiation was found. Downregulation of c-myc and upregulation of p21/Waf1 proteins showed the same time-course in both cell lines. The bcl-2 mRNA was rapidly downregulated while bax and bclxL gene expression was not altered in both differentiating HL60 and HL60/ADR cells. Significant downregulation of bcl-2 protein occurred only in parental HL60 cells. In HL60/ADR, despite rapid cessation of bcl-2 protein synthesis, almost no change in steady-state bcl-2 protein level was found. The lack of bcl-2 protein downregulation was a result of the prolonged half-life of this protein in HL60/ADR cells. Thus, although downregulation of bcl-2 mRNA is coupled to differentiation, actual loss of bcl-2 protein is not required for accomplishment of the differentiation program.

Blotting, Western↗

PubMed1996

Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway.

The serine/threonine kinase Raf-1 functions downstream of Rats in a signal transduction cascade which transmits mitogenic stimuli from the plasma membrane to the nucleus. Raf-1 integrates signals coming from extracellular factors and, in turn, activates its substrate, MEK kinase. MEK activates mitogen-activated protein kinase (MAPK), which phosphorylates other kinases as well as transcription factors. Raf-1 exists in a complex with HSP90 and other proteins. The benzoquinone ansamycin geldanamycin (GA) binds to HSP90 and disrupts the Raf-1-HSP90 multimolecular complex, leading to destabilization of Raf-1. In this study, we examined whether Raf-1 destabilization is sufficient to block the Raf-1-MEK-MAPK signalling pathway and whether GA specifically inactivates the Raf-1 component of this pathway. Using the model system of NIH 3T3 cells stimulated with phorbol 12-myristate 13-acetate (PMA), we show that GA does not affect the ability of protein kinase C alpha to be activated by phorbol esters, but it does block activation of MEK and MAPK. Further, GA does not decrease the activity of constitutively active MEK in transiently transfected cells. Finally, disruption of the Raf-1-MEK-MAPK signalling pathway by GA prevents both the PMA-induced proliferative response and PMA-induced activation of a MAPK-sensitive nuclear transcription factor. Thus, we demonstrate that interaction between HSP90 and Raf-1 is a sine qua non for Raf stability and function as a signal transducer and that the effects observed cannot be attributed to a general impairment of protein kinase function.

3T3 Cells↗

PubMed1995

Possible role for serine/threonine phosphorylation in the regulation of the heteroprotein complex between the hsp90 stress protein and the pp60v-src tyrosine kinase.

The abundant, cytoplasmic 90-kDa heat-shock protein associates transiently with the Rous sarcoma virus oncogenic protein tyrosine kinase, pp60v-src, directs its cellular trafficking and negatively regulates its kinase activity. Here we report that the serine/threonine phosphatase inhibitor, okadaic acid, destabilized the heat-shock protein 90-pp60v-src chaperone complex in v-src-transfected cells. Concomitant with complex destabilization by okadaic acid, phosphoserine was doubled and phosphothreonine was increased 20-fold in the heat-shock protein 90. Although phosphorylation of the total pool of immunoprecipitable pp60v-src was unchanged, okadaic acid slightly increased phosphoserine and phosphothreonine levels specifically in pp60v-src bound to heat-shock protein 90. The low level of tyrosine phosphorylation in the pp60v-src complexed with heat-shock protein 90 was further decreased by okadaic acid. Interestingly, okadaic acid-stabilized hyperphosphorylation of the heat-shock protein 90-pp60v-src complex lowered the level of pp60v-src in cell membranes, the functional location for pp60v-src. We suggest that serine/threonine phosphorylation of heat-shock protein 90 and/or pp60v-src functions as a regulatory molecular trigger to release pp60v-src from the chaperone complex at the inner surface of cell membranes.

3T3 Cells↗

PubMed1995

Bovine serum albumin is a major oligonucleotide-binding protein found on the surface of cultured cells.

To better understand the uptake of oligonucleotides into cells, we have studied the labeling of cell surface proteins by an oligonucleotide conjugated to a radiolabeled photoactivatable crosslinker (Denny-Jaffe reagent). When HL60 cells are treated with the conjugate for 2 hours in a medium containing bovine serum albumin (BSA), almost all of the cell-associated label is found in one protein, which we identify as BSA. Cells grown and treated in a serum-free medium do not show this protein, whereas it is plainly seen in cells that are grown in serum-containing medium but then treated in serum-free medium. Overall association of the oligonucleotide with cells is much higher in serum-free medium than in BSA-containing medium, but the oligonucleotide is mostly not protein-associated in the absence of BSA. We conclude that (1) BSA from the medium serves to block overall association of oligonucleotide with cells, and (2) BSA is the main cell surface protein binding oligonucleotides. We discuss the possible role of albumin in endocytic uptake of oligonucleotides in the cell and in the biodistribution of oligonucleotides in vivo.

Animals↗

PubMed1995

Inhibition of interferon-gamma-induced intercellular adhesion molecule-1 expression on human keratinocytes by phosphorothioate antisense oligodeoxynucleotides is the consequence of antisense-specific and antisense-non-specific effects.

Expression of intercellular adhesion molecule-1 (ICAM-1) by keratinocytes is an important event in the pathogenesis of T-cell-mediated inflammatory skin diseases. To determine if ICAM-1 expression could be selectively modulated, two antisense phosphorothioate oligonucleotides (S-ODN) targeting the translation initiation and 3' untranslated regions of ICAM-1 mRNA were added as lipid complexes to cultures of keratinocytes. Interferon-gamma was added after 24 h to induce ICAM-1 expression, which was quantitated by flow cytometry after 48 h. The S-ODN targeting the translation initiation site did not inhibit ICAM-1 expression at 0.2-20.0 microM. In contrast, 0.2-1.0 microM of the S-ODN targeting a site in the 3' untranslated region abrogated ICAM-1 expression in up to 75% of the keratinocytes; this inhibition was reversible when complementary sense S-ODN was added. Phosphodiester ODN (PD-ODN) targeting the same sites did not inhibit ICAM-1 expression on keratinocytes, most likely as a consequence of rapid degradation. Inhibition of ICAM-1 by the antisense S-ODN was selective; expression of beta 2-microglobulin, alpha 3-integrin, and beta 1-integrin remained largely unaffected and interferon-gamma-induced HLA-DR expression was inhibited to a much lesser extent than ICAM-1. Antisense-non-specific inhibition was also noted in that two scrambled S-ODN with an identical nucleotide (14 of 20 cytosines) composition inhibited ICAM-1 expression in up to 44% of the keratinocytes, whereas a degenerate S-ODN did not. The data demonstrate the complex effects exerted by antisense S-ODN in that ICAM-1 expression was inhibited via antisense-non-specific mechanisms probably due to the intrinsic properties of the S-ODN as well as via the anticipated sequence-specific mechanisms.

Base Sequence↗

PubMed1994

Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation.

The molecular mechanisms by which oncogenic tyrosine kinases induce cellular transformation are unclear. Herbimycin A, geldanamycin, and certain other benzoquinone ansamycins display an unusual capacity to revert tyrosine kinase-induced oncogenic transformation. As an approach to the study of v-src-mediated transformation, we examined ansamycin action in transformed cells and found that drug-induced reversion could be achieved without direct inhibition of src phosphorylating activity. To identify mechanisms other than kinase inhibition for drug-mediated reversion, we prepared a solid phase-immobilized geldanamycin derivative and affinity precipitated the molecular targets with which the drug interacted. In a range of cell lines, immobilized geldanamycin bound elements of a major class of heat shock protein (HSP90) in a stable and pharmacologically specific manner. Consistent with these binding data, we found that soluble geldanamycin and herbimycin A inhibited specifically the formation of a previously described src-HSP90 heteroprotein complex. A related benzoquinone ansamycin that failed to revert transformed cells did not inhibit the formation of this complex. These results demonstrate that HSP participation in multimolecular complex formation is required for src-mediated transformation and can provide a target for drug modulation.

3T3 Cells↗

PubMed1994

Cytostatic and cytotoxic activity of sex steroids against human leukemia cell lines.

We investigated effects of sex steroids and analogs (estradiol, DES, norgestrel, progesterone, medroxyprogesterone, and testosterone) on the proliferation and survival of 10 human leukemia/lymphoma cell lines (HL-60, K562, U937, CEM, KG-1, Jurkat, U266, H929, PA and SUNHL). Micromolar concentrations of sex steroids exerted cytostatic and cytotoxic effects on all cell lines tested, irrespective of their sensitivity to glucocorticoids. The order of potency of sex hormones was: DES > progesterone > or = medroxyprogesterone > testosterone > estradiol >> norgestrel. For progesterone and estradiol, cytostatic effects can be achieved at lower concentrations than cytotoxic effects. The most potent agent, DES, exerted half maximal cytotoxic activity at a median concentration of 4 microM (for 10 leukemia cell lines). Our results provide a basis for the potential therapeutic use of estrogens and progestins in glucocorticoid-resistant leukemias and lymphomas.

Androgens↗

PubMed1994

Oligonucleotides protect cells from the cytotoxicity of several anti-cancer chemotherapeutic drugs.

The possibility of inhibiting gene expression with antisense oligonucleotides (AS ODNs) in combination with more conventional chemotherapy is a very attractive modality in oncology. However, possible interaction between the ODN and drug must be considered. Here we show that ODNs protect cells from the cytostatic/cytotoxic action of actinomycin D (AMD), adriamycin, daunomycin or quinacrine, but not mitomycin, camptothecin, vincristine, cisplatin, etoposide (VP-16) or cycloheximide. The cytoprotective effect depends on ODN length as well as ability to interact directly with the cytotoxic drug and is only slightly sequence selective.

Animals↗

PubMed1993

Stability, clearance, and disposition of intraventricularly administered oligodeoxynucleotides: implications for therapeutic application within the central nervous system.

We report experiments in the rat demonstrating the feasibility of intraventricular administration of oligodeoxynucleotides (ODNs) as a regional treatment approach to disorders within the central nervous system (CNS). Although we find little intrinsic nuclease activity in cerebrospinal fluid (CSF), phosphodiester ODNs are rapidly degraded by brain-associated alpha-exonuclease activity. Phosphorothioate ODNs, however, appear resistant to degradation in the CNS and, after intraventricular administration, we find they are cleared in a manner consistent with CSF bulk flow. Continuous infusion of ODN at 1.5 nmol/hr by miniosmotic pump can maintain micromolar concentrations of intact phosphorothioate ODN in CSF for at least 1 week without obvious neurologic or systemic toxicity. After infusion, extensive brain penetration and marked cellular uptake, especially by astrocytic cells, is demonstrated.

Animals↗

PubMed1993

Theophylline induced differentiation provides direct evidence for the deregulation of c-myc in Burkitt's lymphoma and suggests participation of immunoglobulin enhancer sequences.

Most of the evidence that supports the hypothesis that the c-myc gene is abnormally regulated in Burkitt's lymphoma (BL) is indirect. The putative abnormal expression of c-myc is likely, at least in part, to be a consequence of the usurpation of its regulatory sequences by immunoglobulin enhancer elements, which are invariably juxtaposed to c-myc by the translocations associated with this tumor (C. M. Croce, J. Erikson, A. Ar-Rushdi, D. Aden, and K. Nishikura, Proc. Natl. Acad. Sci. USA, 81: 3170-3174, 1984). We have developed a differentiation induction model system to examine this issue more directly. In a variety of non-BL cell lines, differentiation induction results in the down-regulation of c-myc (G. P. Studzinski, A. K. Bhandal, and Z. S. Brelvi, Proc. Soc. Exp. Biol. Med., 179: 288-295, 1985; Y. Matsui, R. Takahasi, K. Minara, T. Nakagawa, T. Koizumi, Y. Nakao, T. Sugiyama, and T. Fugita, Cancer Res., 49: 1366-1371, 1985; T. Mitchell, E. Sariban, and D. Kufe, Mol. Pharmacol., 30: 398-402, 1986; Z. S. Brelvi, and G. P. Studzinski, J. Cell. Physiol., 128: 171-179, 1986). Since BL is of B-cell origin, differentiation is associated with persistent or increased expression of immunoglobulin genes. Therefore, if c-myc and c-mu are coregulated in BL via immunoglobulin enhancer sequences, persistent or increased expression of the c-myc gene, rather than down-regulation, should occur in differentiated BL cells. Differentiation was induced in four BL cell lines with theophylline (7 x 10(-3) M), and mRNA was examined by Northern blot analysis. In all four BL lines studied (JD38, AG876, KK124, and Daudi), there was persistent or increased expression of both c-mu and c-myc genes (detected with a third exon c-myc probe), in contrast to the decreased expression of the c-myc gene observed in the three Epstein-Barr virus transformed lines studied (A3317, TC84, and CB34). In the BL cell line, JD38, the c-myc gene is truncated (the second and third exons are translocated to chromosome 14 while the first exon remains on chromosome 8). In this line, we demonstrated that theophylline induced differentiation results in down-regulation of the first exon while the level of expression of the translocated second and third exons remains unchanged or increases.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Benzoquinonoid ansamycins possess selective tumoricidal activity unrelated to src kinase inhibition.

The benzoquinonoid ansamycin antibiotics herbimycin A and geldanamycin have been shown to reverse the oncogenic phenotype of pp60v-src transformed cells as well as induce differentiation in a number of in vitro model systems, reportedly due to their inhibition of src family protein tyrosine kinases. We now report that these agents are potent cytotoxins in vitro against a panel of highly malignant human tumor cell lines possessing primitive neural features. Proliferation and/or survival of fibroblasts, primary neuronal cultures, and several leukemia cell lines are unaffected at concentrations resulting in greater than 99% cell loss in sensitive lines. The tumorigenicity in nude mice of sensitive cell lines can also be markedly reduced by either systemic or topical administration of these agents without apparent toxicity to the whole animal. The cytocidal action of these ansamycins is initiated very rapidly, is irreversible, and is clearly distinct from the delayed inhibition of src family kinases that has been reported previously. Due to their potency, relative selectivity, and novel mechanism(s) of action, these drugs could prove clinically useful in the therapy of a number of human cancers of neural derivation.

3T3 Cells↗

PubMed1992

Analysis of oligonucleotide binding, internalization, and intracellular trafficking utilizing a novel radiolabeled crosslinker.

Although antisense oligonucleotides have been widely used to inhibit gene expression, their mechanism of entry into cells and their site of action are still in some doubt. In this report, we describe a novel technique for kinetically analyzing oligonucleotide association with living cells as well as intracellular compartmentalization. The technique utilizes a photoactivatable, radiolabeled crosslinker, the Denny-Jaffe reagent. Oligonucleotides containing pendant amine groups were conjugated to this reagent, added to HL60 cells in culture, and photocrosslinked to associated proteins, which were analyzed electrophoretically. We find that several proteins are labeled, predominantly a 75 kD one that appears to be membrane-associated. Our results suggest that the majority of intracellular oligonucleotide is associated in vesicles with the same protein to which it bound on the cell surface, but only a small percentage of non-protein-bound cytosolic oligonucleotide can be detected. Additionally, oligonucleotides are readily accumulated by nuclei, and by treating whole nuclei, a unique set of nuclear binding proteins is detected.

Azides↗

PubMed1992

Incorporation of radiophosphorus from labeled oligodeoxynucleotides into RNA of mycoplasma in cell cultures.

We have found that various mycoplasma species quickly and efficiently incorporate radiophosphorus into their RNA from labeled oligonucleotides added to the medium. The label can be in any of several positions in an oligodeoxynucleotide, and incorporation also occurs efficiently from labeled RNA. Mycoplasmas also incorporate the radiolabel when they infect a mammalian cell culture; the host cells do not. This incorporation presumably involves uptake of the oligodeoxynucleotide followed by digestion to mononucleotides, conversion to ribonucleotides, and incorporation in new RNA. We believe that the processing of oligodeoxynucleotides by mycoplasma could be a source of artifacts in antisense work in cell culture and could have implications for the development of antisense therapeutics. We also suggest ways to exploit the incorporation phenomenon in mycoplasma testing.

Adenosine Triphosphate↗

PubMed1992

Antisense inhibition of gene expression: a tool for studying the role of NMYC in the growth and differentiation of neuroectoderm-derived cells.

To understand the role of individual genes in regulating biological processes, one must be able to interfere specifically with either their expression or function. While monoclonal antibodies have proven very useful in studying cell surface proteins, the specific inhibition of intracellular proteins in viable cells is a much more difficult problem. The goal of antisense technology is to develop small oligonucleotides, plasmids, or retroviral vectors that can be introduced easily into viable cells in order to inhibit gene products specifically. In this report, we will describe our use of antisense DNA and RNA to study the role of the NMYC proto-oncogene in neuroectodermal cell growth and differentiation.

Animals↗

PubMed1992

A phase I study of interleukin-2 in children with cancer.

Recombinant interleukin-2 (IL-2) produces clinical responses in approximately 20% of adult patients with renal cell carcinoma and melanoma, with both high-dose bolus and continuous infusion regimens. Because of the lower toxicity of continuous infusion, we elected to investigate in a Phase I trial a 5-day continuous infusion repeated for three weeks in children with malignancies refractory to standard therapy. Nineteen children with solid tumors and eight children with hematologic malignancies were entered into the study. The maximum tolerated dose was 3 x 10(6) U/m2/day, with dose-limiting toxicities occurring in five of seven patients treated at the 5 x 10(6) U/m2/day dose level. Dose-limiting toxicities included hypotension, hyperbilirubinemia, thrombocytopenia, pulmonary/pleural effusion, and nephrotoxicity. Serum IL-2 levels were detectable at the higher dose levels and were comparable to those observed in adult patients. Hematologic changes at the higher dose levels included rebound lymphocytosis occurring within 48 h of discontinuation of IL-2, eosinophilia, and decreased platelet counts. No objective responses to therapy were seen. We have identified a dose and schedule of administration for IL-2 in pediatric patients that can be given without intensive care unit support. Pediatric Phase II trials examining the anti-tumor activity of IL-2 given by this schedule are in progress.

Adolescent↗