Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals.

Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.

Derivation of human tumor cells in vitro without widespread genomic instability.

The majority of adult human epithelial cancers exhibit evidence of genetic instability, and it is widely believed that the genetic instability manifested by aneuploidy or microsatellite instability plays an essential role in the genesis of these tumors. Indeed, most experimental models of cancer also show evidence of genomic instability. The resulting genetic chaos, which has widespread effects on many genes throughout the genome, confounds attempts to determine the precise cohort of genetic changes that are required for the transformation of normal human cells to a tumorigenic state. Here we show that genetic transformation of human kidney epithelial cells can occur in the absence of extensive aneuploidy, chromosomal translocations, and microsatellite instability. These observations demonstrate that the in vitro oncogenic transformation of human cells can proceed without widespread genomic instability.

TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.

Inhibition of telomerase limits the growth of human cancer cells.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

Creation of human tumour cells with defined genetic elements.

During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.

Determination of carbosulfan in oranges by high-performance liquid chromatography with post-column fluorescence.

High-performance liquid chromatography equipped with post-column fluorescence derivatization provides an automated and specific analytical method for pesticide determination. Carbosulfan, a carbamate pesticide, can be analysed utilizing an existing and proven carbamate system after the addition of one additional hydrolytic reactor in-line. Extraction of this compound from citris crops requires careful handling owing to the volatility and unstable nature of the analyte in acid conditions. This paper describes the necessary instrument modifications and presents a method for carbosulfan extraction. Analyses of orange homogenates were performed rapidly with recoveries of between 66 and 98% for residue levels between 0.03 and 0.25 micrograms kg-1.

Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation.

The proteins Grb2-Sem-5, Shc and Sos have been implicated in the signalling pathway from tyrosine kinase receptors to Ras. Grb2-Sem-5 binds directly to murine Sos1, a Ras exchange factor, through two SH3 domains. Sos is also associated with ligand-activated tyrosine kinase receptors which bind Grb2-Sem-5, and with the Grb2-Sem-5 binding protein, Shc. Ectopic expression of Drosophila Sos stimulates morphological transformation of rodent fibroblasts. These data define a pathway by which tyrosine kinases act through Ras to control cell growth and differentiation.

Oncogenic activity of the c-Myc protein requires dimerization with Max.

c-Myc (Myc) and Max proteins dimerize and bind DNA through basic-helix-loop-helix-leucine zipper motifs (b-HLH-LZ). Using a genetic approach, we demonstrate that binding to Max is essential for Myc transforming activity and that Myc homodimers are inactive. Mutants of Myc and Max that bind efficiently to each other but not to their wild-type partners were generated by either exchanging the HLH-LZ domains or reciprocally modifying LZ dimerization specificities. While transformation defective on their own, complementary mutants restore Myc transforming activity when coexpressed in cells. The HLH-LZ exchange mutants also have dominant negative activity on wild-type Myc function. In addition, wild-type max antagonizes myc function in a dose-dependent manner, presumably through competition of Max-Max and Myc-Max dimers for common target DNA sites. Therefore, Max can function as both suppressor and activator of Myc. A general model for the role of Myc and Max in growth control is discussed.

Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max.

The c-myc protein (Myc) contains an amino-terminal transcriptional activation domain and a carboxy-terminal basic helix-loop-helix-leucine zipper (bHLH-Z) domain that directs dimerization of Myc with its partner, the max protein (Max), and promotes DNA binding to sites containing a CACGTG core consensus sequence. Despite these characteristics and the observation that Myc can modulate gene expression, a direct role for Myc or Max as transcription factors has never been demonstrated. Here we use Saccharomyces cerevisiae as an in vivo model system to show that the Myc protein is a sequence-specific transcriptional activator whose DNA binding is strictly dependent on dimerization with Max. Transactivation is mediated by the amino-terminal domain of Myc. We find that Max homodimers bind to the same DNA sequence as Myc+Max but that they fail to transactivate and thus can antagonize Myc+Max function. We also show that the Max HLH-Z domain has a higher affinity for the Myc HLH-Z domain than for itself, and suggest that the heterodimeric Myc+Max activator forms preferentially at equilibrium.

Domains of human c-myc protein required for autosuppression and cooperation with ras oncogenes are overlapping.

Amino acids 106 to 143 and 354 to 433 of the human c-myc protein (439 amino acids) were shown to be required for the protein to suppress c-myc gene transcription and were found to exactly overlap with those necessary for c-myc to cooperate with ras oncogenes in the transformation of rat embryo fibroblasts. The essential carboxyl-terminal region harbors structural motifs (a basic region, a helix-loop-helix motif, and a "leucine zipper"), which, in other proteins, can mediate dimerization and sequence-specific DNA binding.

Negative autoregulation of c-myc transcription.

The introduction of activated c-myc and v-myc genes into a variety of non-established and established cells results in the suppression of endogenous c-myc expression. As measured in Rat-1 fibroblasts, the suppression occurs at the level of transcriptional initiation. Moreover, the extent of the down-regulation is proportional to the cellular concentration of c-myc protein, and the critical concentration range in which the endogenous c-myc RNA is effectively suppressed corresponds to that found in non-transformed cells. In addition, the autoregulatory mechanism is not only dependent on c-myc protein, but also requires additional trans-acting factors. These results support a role for c-myc in the regulation of cellular gene transcription and suggest that a negative feedback mechanism can act as a homeostatic regulator of c-myc expression in vivo.

Role of ion channels and intraterminal calcium homeostasis in the action of deltamethrin at presynaptic nerve terminals.

Using a continuous perfusion system, synaptosomes prepared from rat brain released [3H]norepinephrine in a Ca2+-dependent manner when pulse depolarized by briefly elevating external potassium concentrations. Tetrodotoxin (10(-7) M), a sodium channel blocker, inhibited 48% of this pulsed release, and D595 (10(-5) M), a phenethylamine-type calcium channel blocker, inhibited 21%. In combination, these two specific ion channel antagonists appear to function independently of each other in an additive fashion. Addition of deltamethrin to this preparation resulted in an enhanced release of [3H]norepinephrine which occurred in a biphasic fashion. At 10(-7) M, deltamethrin produced a 42% enhancement in the first or initial peak of [3H]norepinephrine release and a 100% enhancement in the second or tailing peak. Addition of deltamethrin to tetrodotoxin-pretreated synaptosomes resulted in a net 37% enhancement of the initial peak release and a net increase of 277% in the tailing peak. Addition of deltamethrin to D595-pretreated synaptosomes produced no significant effect on enhanced [3H]norepinephrine release from either peak. Since tetrodotoxin is a specific sodium channel blocker, deltamethrin may be enhancing [3H]norepinephrine release by increasing the uptake of Ca2 via other voltage-gated channels (e.g. calcium) or exchange mechanisms in addition to its action at voltage-gated sodium channels. To determine whether deltamethrin may also have an effect on intraterminal Ca2+ homeostasis, external Ca2+ was replaced with Ba2+ and synaptosomes were depolarized with pentylenetetrazole (PTZ). At 10(-5) M, deltamethrin produced a 66% increase in neurotransmitter release over that produced by PTZ alone. An estimated EC50 value of deltamethrin for PTZ-induced release was calculated to be 2.4 x 10(-10) M.

Rapid method for the determination of alachlor, atrazine and metolachlor in groundwater by solid-phase extraction.

A solid-phase extraction method is described for the separation of alachlor, atrazine and metolachlor from groundwater using solid-phase disposable columns. The method is rapid, reproducible and uses considerably fewer reagents than classical liquid-liquid methods. The average recoveries were greater than 90% for all three compounds.