Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.

Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC.

Induction of multiple programmed cell death pathways by IFN-beta in human non-small-cell lung cancer cell lines.

Tissue transglutaminase (tTG) and keratinocyte transglutaminase (kTG), as well as the cross-linked envelopes (CLE) that they form, have been associated with squamous differentiation and programmed cell death in epithelial cells. When interferon-beta (IFN-beta) was used to stimulate differentiation and programmed cell death in the human lung cancer cell lines NCI-H596 and NCI-H226, the cells underwent a decrease in cellular density. In NCI-H596 IFN-beta caused an increase in kTG activity and DNA fragmentation in the lower density cells, which were significantly slower growing than control cells. However, in the higher density cells, which were only slightly slower growing than control cells, IFN-beta caused an increase in tTG activity and CLE competence. Dual-parameter flow cytometry demonstrated that IFN-beta-induced squamous differentiation preceded programmed cell death. Treatment of NCI-H596 cells with monodansylcadaverine, a transglutaminase inhibitor, prevented the increase in CLE competence, but did not inhibit DNA fragmentation. These results suggest that IFN-beta can induce NCI-H596 cells to enter multiple cell death pathways and that these pathways are not only differentiation related, but may also be growth driven.

Tyrosine kinase inhibitors in preclinical development.

Due to the limited efficacy of cytotoxic chemotherapy in the treatment of advanced malignancy and its excessive toxicity precluding its use in chemoprevention, new therapeutic and preventive strategies have been sought. One of the most interesting of these new approaches is the manipulation of signal transduction pathways. Among the approaches being considered to eventuate such a strategy is the inhibition of autophosphorylation, a critical first step in the signal transduction pathways of many cell surface receptor tyrosine kinases, as well as of non-receptor tyrosine kinases. This article is intended to review those tyrosine kinase inhibitors that are currently in preclinical development, for which there are data to support consideration for their use in chemoprevention or cancer treatment. We will focus upon those agents that have received attention in the past several years.

Myotonia and the muscle chloride channel: dominant mutations show variable penetrance and founder effect.

The delayed relaxation or sustained contraction of skeletal muscle-myotonia-is frequently seen in myotonic dystrophy and sodium channelopathies (hyperkalemic periodic paralysis, paramyotonia congenita). Many cases of congenital myotonia without other clinical symptoms have been associated with mutations in the muscle chloride channel gene. Most cases reported to date show a recessive inheritance pattern, with loss of function of the corresponding protein. Six families have been reported with dominantly inherited myotonia and mutations of the chloride channel gene. Here we report clinical and molecular data on 38 family members from four new families with dominantly inherited myotonia congenita. Three families show a previously characterized G230E mutation, and we show that these three share a common affected ancestor despite living in different regions of the United States (linkage disequilibrium). One Italian family is shown to have a novel dominant mutation-I290M. This is the sixth mutation identified in Thomsen's myotonia. Genotype/phenotype correlations in these four families showed that both of the dominant mutations resulted in a mild clinical picture in 90% of the patients, and no symptoms in 10% of mutation-positive patients. The EMG was the clinical feature that most closely correlated with mutation data; however, 3 of 16 (19%) mutation-positive patients tested negative by electromyography at least once, and 1 (6%) tested negative despite multiple tests. Only about half (55%) of the mutation-positive patients tested positive for percussion myotonia. Most of the clinically symptomatic individuals stated that cold temperatures and stress substantially worsened their myotonia. Our data show that dominantly inherited Thomsen's myotonia is most often a very mild disorder that shows considerable clinical heterogeneity.

Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene.

Nitric oxide, a multifunctional effector molecule synthesized by nitric oxide synthase (NOS) from L-arginine, conveys signals for vasorelaxation, neurotransmission, and cytotoxicity. Three different NOS isoforms have been identified which fall into two distinct types, constitutive and inducible. The inducible NOS (iNOS) isoform is expressed in a variety of cell types and tissues in response to inflammatory agents and cytokines. The human iNOS (NOS2) gene was isolated on overlapping cosmid clones from a human genomic library using both the murine macrophage and the human hepatocyte iNOS cDNAs as probes. All isolated cosmids were part of a single genomic locus and no other genomic loci were identified or isolated. Analysis of this locus indicated that the human iNOS gene is approximately 37 kilobases in length and consists of 26 exons and 25 introns. Primer extension analysis of lipopolysaccharide and cytokine-stimulated human hepatocyte RNA mapped the transcriptional initiation site 30 base pairs downstream of a TATA sequence, and a 400-base pair 5'-flanking region was found to be structurally similar to the recently described murine iNOS promoter. Polymerase chain reaction analysis of a human/rodent genomic DNA somatic cell hybrid panel and fluorescent in situ hybridization indicated that the human iNOS gene is located on chromosome 17 at position 17cen-q11.2.