Chelators as Antineuroblastomas Agents.

Neuroblastoma represents 8-10 % of all malignant tumors in childhood and is responsible for 15 % of cancer deaths in the pediatric population. Aggressive neuroblastomas are often resistant to chemotherapy. Canonically, neuroblastomas can be classified according to the MYCN (N-myc proto-oncogene protein) gene amplification, a common marker of tumor aggressiveness and poor prognosis. It has been found that certain compounds with chelating properties may show anticancer activity, but there is little evidence for the effect of chelators on neuroblastoma. The effect of new chelators characterized by the same functional group, designated as HLZ (1-hydrazino phthalazine), on proliferation (WST-1 and methylene blue assay), cell cycle (flow cytometry), apoptosis (proliferation assay after use of specific pharmacological inhibitors and western blot analysis) and ROS production (fluorometric assay based on dichlorofluorescein diacetate metabolism) was studied in three neuroblastoma cell lines with different levels of MYCN amplification. The molecules were effective only on MYCN-non-amplified cells in which they arrested the cell cycle in the G0/G1 phase. We investigated the mechanism of action and identified the activation of cell signaling that involves protein kinase C.

Synthesis and deposition of a Tröger's base polymer on the electrode surface for potentiometric detection of a neuroblastoma tumor marker metabolite.

The development of novel diagnostic tools is a primary goal in bioanalytical chemistry. Here we report the synthesis of Tröger's base functionalized with amino- and coumarin-units designed as a monomeric unit for the development of an electrochemical cancer sensor. The synthesized receptor was deposited onto a conducting support using electrochemical polymerization, characterized spectroscopically and tested potentiometrically towards metabolites used as tumor markers of neuroblastoma.

[Personal experience with laparoscopic treatment of sterility]. / Nase zkusenosti s laparoskopickou lécbou sterility.

OBJECTIVE: Evaluation of laparoscopy in treatment of sterility. DESIGN: Retrospective clinical study. SETTING: Department of Gynaecology and Obstetrics, Hospital in Bruntal, Czech Republic. METHODS: In 1997 and 1998 we performed 36 laparoscopic operations in patients with primary or secondary sterility at our department. In women with anovulatory cycles we performed drilling of the ovaries. Adhesions both in and beyond the pelvis were treated by lysis of adhesions, in women under 35 with mild or moderate hydrosalpinx we performed terminal salpingostomy while large hydrosalpinges were treated with salpingectomy and the patients were recommended for assisted reproduction. Success of the treatment was evaluated at least half a year after laparoscopy. RESULTS: In the anovulatory group, 60% conceived on average 2 months after operation. 36.4% conceived in the group with pelvic adhesions, on average in 5 months and 50% in the salpingostomy group conceived in 8 months. CONCLUSIONS: Although the number of patients is small, the obtained results prove the usefulness of operative laparoscopy in the treatment algorithm of sterile patients.

Activation of Bruton's tyrosine kinase (BTK) by a point mutation in its pleckstrin homology (PH) domain.

Bruton's tyrosine kinase (BTK) is a nonreceptor tyrosine kinase critical for B cell development and function. Mutations in BTK result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Using a random mutagenesis scheme, we isolated a gain-of-function mutant called BTK* whose expression drives growth of NIH 3T3 cells in soft agar. BTK* results from a single point mutation in the pleckstrin homology (PH) domain, where a Glu is replaced by Lys at residue 41. BTK* shows an increase in phosphorylation on tyrosine residues and an increase in membrane targeting. Transforming activity requires kinase activity, a putative autophosphorylation site, and a functional PH domain. Mutation of the SH2 or SH3 domains did not affect the activity of BTK*. Expression of BTK* could also relieve IL-5 dependence of a B lineage cell line. These results show that transformation activation and regulation of BTK are critically dependent on the PH domain.

The nuclear tyrosine kinase c-Abl negatively regulates cell growth.

c-Abl is a tyrosine kinase localized primarily in the nucleus. Previous assays for abl function rely on cellular transformation by abl mutants, which are cytoplasmic. Using a conditional overexpression strategy, we have developed a functional assay for c-abl. Overexpression of c-abl inhibits growth by causing cell cycle arrest. Growth suppression requires tyrosine kinase activity, nuclear localization, and an intact SH2 domain. Overexpression of dominant negative c-abl disrupts cell cycle control and enhances transformation by tyrosine kinases, G proteins, and transcription factor oncogenes. These findings suggest that c-abl acts as a negative regulator of cell growth. This growth suppressive activity is functionally similar to that of tumor suppressor genes such as p53 and Rb.

En bloc substitution of the Src homology region 2 domain activates the transforming potential of the c-Abl protein tyrosine kinase.

Src homology region 2 (SH2) domains are present in many proteins involved in signal transduction. In nonreceptor protein tyrosine kinases the SH2 domain has been implicated in regulation of tyrosine kinase activity and in mediating interactions involved in downstream signaling. Different SH2 domains exhibit distinct binding specificities for both phosphotyrosine- and phosphoserine/phosphothreonine-containing proteins. We show that different SH2 domains are not functionally equivalent within the context of the c-ABL1b protooncogene. c-ABL1b, altered by replacement of its SH2 domain with the N-terminal SH2 domain of Ras GTPase-activating protein, exhibited activated transforming capability, caused intracellular tyrosine phosphorylation of p62, and was relocalized from nucleus to cytoplasm. This en bloc substitution apparently uncouples two distinct functions of the SH2 domain so that c-ABL escapes normal regulatory control while it retains the capability to elicit signals that promote transformation. The SH2 domain of the ARG protein tyrosine kinase, which shares high amino acid-sequence homology with the SH2 domain of ABL, was less effective in activating the oncogenic potential of c-ABL. The effects that the N-terminal SH2 domain of Ras GTPase-activating protein has in the context of c-ABL resemble the effects of deleting the SH3 domain. Thus, the SH2 and SH3 domains may have coordinate roles as regulatory control elements within the context of c-ABL.

SH1 domain autophosphorylation of P210 BCR/ABL is required for transformation but not growth factor independence.

P210 BCR/ABL is a chimeric oncogene implicated in the pathogenesis of chronic myelogenous leukemia. BCR sequences have been shown to be required for activation of the tyrosine kinase and transforming functions of BCR/ABL. In this work, we show that two other structural requirements for full transforming activity of P210 BCR/ABL include a functional tyrosine kinase and the presence of tyrosine 1294, a site of autophosphorylation within the tyrosine kinase domain. Replacement of tyrosine 1294 with phenylalanine (1294F) greatly diminishes the transforming activity of BCR/ABL without affecting the specific activity of the protein tyrosine kinase. Expression of an exogenous myc gene in fibroblasts partially complements the transforming capacity of mutant P210 BCR/ABL (1294F). Surprisingly, tyrosine 1294 is not required for efficient induction of growth factor-independence in hematopoietic cell lines by P210 BCR/ABL. These results suggest that autophosphorylation at tyrosine 1294 may be important for recognition and phosphorylation of cellular substrates in the pathway of transformation, but it is not critical for mediating the events which lead to growth factor independence.

A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction.

SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.

BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner.

BCR-ABL is a chimeric oncogene implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL. We have tested the hypothesis that activation of BCR-ABL may involve direct interaction between BCR sequences and the tyrosine kinase regulatory domains of ABL. Full-length c-BCR as well as BCR sequences retained in BCR-ABL bind specifically to the SH2 domain of ABL. The binding domain has been localized within the first exon of BCR and consists of at least two SH2-binding sites. This domain is essential for BCR-ABL-mediated transformation. Phosphoserine/phosphothreonine but not phosphotyrosine residues on BCR are required for interaction with the ABL SH2 domain. These findings extend the range of potential SH2-protein interactions in growth control pathways and suggest a function for SH2 domains in the activation of the BCR-ABL oncogene as well as a role for BCR in cellular signaling pathways.

Evidence for regulation of the human ABL tyrosine kinase by a cellular inhibitor.

Phosphotyrosine cannot be detected on normal human ABL protein-tyrosine kinases, but activated oncogenic forms of the human ABL protein are phosphorylated on tyrosine in vivo. Activation of ABL can occur by substitution of the ABL first exon with breakpoint cluster region (BCR) sequences or by deletion of the noncatalytic SH3 (src homology region 3) domain. An alternative mode for the activation of the ABL kinases is hyperexpression at greater than 500-fold over endogenous levels. This is not a consequence of transphosphorylation of the hyperexpressed ABL molecules. ABL proteins translated in vitro lack phosphotyrosine, but tyrosine kinase activity is uncovered after immunoprecipitation and removal of lysate components. The rates of dephosphorylation of ABL and BCR-ABL fusion protein by phosphotyrosine-specific phosphatases are approximately the same. These combined results indicate that inhibition of ABL activity is reversible and suggest that a cellular component interacts noncovalently with ABL to inhibit its autophosphorylation.

The mitochondrial genome organization of a maize fertile cmsT revertant line is generated through recombination between two sets of repeats.

The mitochondrial genome (mtDNA) organization from a fertile revertant line (V3) derived from the maize cytoplasmic male sterile type T (cmsT) callus tissue culture has been determined. We report that the sequence complexity can be mapped on to a circular "master chromosome" of 705 kb which includes a duplication of 165 kb of DNA when compared to its male sterile progenitor. Associated with this event is also a 0.423-kb deletion, which removed the cmsT-associated urf13 gene. As found for the maize normal type (N) and cmsT mitochondrial genomes, the V3 master chromosome also exists as a multipartite structure generated by recombination through repeated sequences.

Study of two different recombination events in maize cmsT-regenerated plants during reversion to fertility.

The change of phenotype from sterility to fertility for some cmsT callus tissue culture regenerated plants and their progenies has been correlated with changes in their mitochondrial genome. Those changes that have been analyzed here are the result of recombination events. Two different sets of repeated sequences have been found to be involved in those recombination events. The most common one is a recombination through a 127-bp repeat between various independently isolated revertants. The second one is a recombination through a 58-bp repeat. In every case the products of recombination containing the urf13 gene have been deleted.

Chimeric organization of two genes for the soybean mitochondrial ATPase subunit 6.

There are two copies of the ATPase subunit 6 (atp6) gene in the soybean mitochondrial genome which differ in their gene organization but share extensive homology with the maize atp6 gene except at their 5' ends. The two soybean genes are chimeric, containing regions with homology to other known mitochondrial genes at their 5' ends. Sequences homologous to the cytochrome oxidase subunit II (coxII) are located in one copy and sequences homologous to the ATPase subunit 9 (atp9) gene are located in the other copy, both of which contain methionine (ATG) codons that are in-frame with the remainder of the atp6 open reading frame. At least the copy of atp6 that contains the coxII sequence at its 5' end is abundantly transcribed to give an RNA of approximately 1,200 nucleotides.