Protein farnesylation in mammalian cells: effects of farnesyltransferase inhibitors on cancer cells.

Protein farnesylation, catalyzed by protein farnesyltransferase, plays important roles in the membrane association and protein-protein interaction of a number of eukaryotic proteins. Recent development of farnesyltransferase inhibitors (FTIs) has led to further insight into the biological significance of farnesylation in cancer cells. A number of reports point to the dramatic effects FTIs exert on cancer cells. In addition to inhibiting anchorage-independent growth, FTIs cause changes in the cell cycle either at the G1/S or at the G2/M phase. Furthermore, induction of apoptosis by FTIs has been reported. FTIs also affects the actin cytoskeleton and cell morphology. This review summarizes these reports and discusses implications for farnesylated proteins responsible for these FTI effects.

Potentiation of nitric oxide-induced apoptosis of MDA-MB-468 cells by farnesyltransferase inhibitor: implications in breast cancer.

High amounts of nitric oxide (NO) produced by activated macrophages or NO donors are required to induce cytotoxicity and apoptosis in pathogens and tumor cells. High concentrations of NO may lead to nonspecific toxicity thereby limiting the use of NO donors in the treatment of cancer. In this study, we tested the possibility of potentiating the apoptotic action of NO in a human breast cancer cell line, MDA-MB-468, by combining it with a farnesyltransferase inhibitor (FTI), which has been shown to induce apoptosis in some other cancer cell lines with minimal toxicity to normal cells. DETA-NONOate, a long acting NO donor which has a half-life of 20 h at 37 degrees C, was used in this study. DETA-NONOate (1 mM), which releases NO in the range produced by activated macrophages, induced apoptosis after 36 h in MDA-MB-468 cells via cytochrome c release and caspase-9 and -3 activation. FTI (25 microM) potentiated the action of lower concentrations of DETA-NONOate (25-100 microM) by inducing apoptosis in these cells within 24 h by increasing cytochrome c release and caspase-9 and -3 activation. This effect was observed preferentially in the cancer cell lines studied with no apoptosis induction in normal breast epithelial cells. This novel combination of FTI and NO may emerge as a promising approach for the treatment of breast cancer.

Changes in permeability of strial vessels following vibration given to auditory ossicle by drill.

We produced drill-induced damage of the auditory ossicles of guinea pigs to study changes over time in the permeability of the blood vessels of the stria vascularis to horseradish peroxidase (HRP). In group A, the stimulus was applied for 10 seconds after intravenous injection of HRP. In group B, it was applied for 30 seconds, and in group C, for 60 seconds. The cochlea was fixed with 2% glutaraldehyde perfused through the round window, and the guinea pigs were then decapitated. The stria vascularis of the basal and third turns was examined. The leakage of HRP from the blood vessels of the stria vascularis significantly increased in relation to the duration of the stimulus in both the basal and third turns. The damage to intermediate cells also tended to be in relation to the duration of the stimulus. Extravascular permeation of HRP took place through the tubules in the endothelial cytoplasm. The vibratory stimulation presumably opened channels that are not normally open.

LUCA15, a putative tumour suppressor gene encoding an RNA-binding nuclear protein, is down-regulated in ras-transformed Rat-1 cells.

BACKGROUND: The proliferation of mammalian cells is controlled by various intracellular mitogenic signalling pathways. In the intracellular pathways, Ras is involved in the activation of proto-oncogenes such as an immediate early gene c-fos. The somatic mutations of ras genes that elicit the constitutive activation of Ras have been found in tumours. Although these findings suggest that the constitutive activation of Ras-mediated pathways alters the expression of a set of genes involving tumorigenesis, these genes have not yet fully been studied. RESULTS: To study the up- or down-regulated genes in ras-transformed cells, we analysed Rat-1 transfectants expressing Ras(G12V) mutant protein in response to isopropyl-1-beta-thio-D-galactoside using a differential display. We found that the mRNA level of rat homologue of LUCA15, which has been cloned initially as a putative tumour suppressor gene mapped on human chromosome 3, was down-regulated by the expression of Ras(G12V). Epitope-tagged LUCA15 protein was localized in nuclei and had the ability to bind poly(G) RNA homopolymers in vitro. Moreover, ectopic expression of LUCA15 in human fibrosarcoma HT1080 cells suppressed the cell growth. CONCLUSION: These results demonstrate that LUCA15 is one of the down-regulated genes in ras-transformed cells, and suggests that LUCA15 may function as a negative regulator of cell proliferation by the alteration of its mRNA level.

Cdk inhibitors, roscovitine and olomoucine, synergize with farnesyltransferase inhibitor (FTI) to induce efficient apoptosis of human cancer cell lines.

Farnesyltransferase inhibitor (FTI) induces apoptosis of transformed cells. This involves changes in mitochondria, including decrease of mitochondrial membrane potential and the release of cytochrome c. The released cytochrome c then induces events leading to the activation of caspase-3. In this study, we report that purine derivative cyclin-dependent kinase (Cdk) inhibitors, roscovitine and olomoucine, dramatically enhance this FTI-induced apoptosis of human cancer cell lines. We noticed the synergy between Cdk inhibitors and FTI through our screen to identify compounds that enhance FTI-induced apoptosis of promyelocytic leukemic cell line HL-60. The Cdk inhibitors by themselves do not induce apoptosis at the concentrations used. Roscovitine synergizes with FTI to release cytochrome c from mitochondria. In addition, we detected synergistic effects of FTI and roscovitine to inhibit hyperphosphorylation of retinoblastoma protein. Enhancement of FTI-induced apoptosis by roscovitine is not unique to HL-60 cells, since similar synergy was observed with a leukemic cell line CEM and a prostate cancer cell line LNCaP. In LNCaP cells, in addition to roscovitine and olomoucine, phophatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, was effective in enhancing FTI-induced apoptosis. However, the effects of roscovitine appear to be distinct from those of LY294002, since roscovitine did not affect Akt activity while LY294002 significantly decreased the activity of Akt. Our finding of the synergy between FTI and Cdk inhibitor is significant for understanding the mechanism of action of FTI as well as for clinical use of FTI.

Expression of an oncogenic mutant G alpha i2 activates Ras in Rat-1 fibroblast cells.

It has been reported that expression of the active mutant of heterotrimeric GTP-binding protein alpha subunit G alpha i2 transforms Rat-1 cells. However, the G alpha i2-mediated mitogenic signaling pathways remain to be elucidated. Here, we demonstrate that inducible expression of the active mutant of G alpha i2 (G alpha i2(Q205L)) activates Ras and c-Jun N-terminal kinase (JNK) in addition to extracellular signal-regulated kinase (ERK) in Rat-1 cells. Our findings suggest that Ras may play a critical role in the G alpha i2-induced transformation and G alpha i2 can transduce signals from the Gi-coupled receptor to JNK and ERK in certain types of mammalian cells.

Activation of Stat1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon-gamma-induced MAPK activation that is mediated by p21ras.

Rat C6 glioma cells have been used to characterize molecular events involved in the regulation of inducible nitric oxide synthase (iNOS) gene expression stimulated by interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). IFNs induce a signaling event which involves activation of Stat1 transcription factor. Previous studies have shown that IFNs also induce extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activation. However, the mechanisms by which IFNs stimulate MAPK activation remain elusive. Here we show that in C6 glioma cells, transiently expressing the dominant-negative form of c-Ha-Ras (Asn-17) abrogated IFN-gamma-induced ERK1 and ERK2 activation. Furthermore, PD98059, a specific MEK1 inhibitor, also blocked this activation. These results indicate that p21ras and MEK1 are required for IFN-gamma-induced ERK1 and ERK2 activation. Recent studies have reported that MAPK is responsible for serine phosphorylation of Stat1 which is required for Stat1's DNA binding and maximal transcriptional activity. Thus, we examined the role of the Ras-MAPK pathway in Stat1 activation and subsequent iNOS induction in C6 glioma cells. Further experiments showed that neither Asn-17 Ras expression nor concentrations of PD98059, which completely abrogated IFN-gamma-induced ERK1 and ERK2 activation, affected Stat1 DNA binding activity or iNOS induction, indicating that the Ras-MAPK pathway does not appear to be involved in the activation of Stat1 and subsequent iNOS induction in C6 glioma cells.

Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1alpha promoter and lac operator.

We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1alpha promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl beta-D-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

[Three-dimensional CT of the ossicles of the middle ear].

This study was performed to evaluate the usefulness and limitations of three-dimensional (3-D) imaging of the ossicular chain in the middle ear by high speed helical CT. One dissected human temporal bone, five normal ears, and twelve diseased ears (trauma, ossicular anomaly, cholesteatoma, chronic otitis media) were scanned in 1.0mm slices and reconstructed at a thickness of 0.2-0.5mm. All 3-D CT specimens can be observed in any plane and from any direction. Ossicular 3-D CT temporal bone images were reconstructed as if the malleus, incus and stapes were being observed under a microscope. No defect in the ossicles or their joints was seen in the images. The entire structure of the stapes could not be represented by conventional two-dimensional CT, but the 3-D CT in our study showed the head, crus and foot plate of the stapes in detail. Ossicular 3-D CT images of normal ears yielded the same findings as those recorded in the temporal bone. Preoperative diagnostic findings of ossicles in diseased ears were very useful. 3-D CT was diagnostic and its accuracy was confirmed by surgical observations, especially in ossicular anomalies. 3-D CT was also an important method of postoperative evaluation of ossicular reconstruction, i.e. TORP and PORP. It could represent the anatomical relation between prosthesis and the oval window. Postoperative hearing improvement can be compared with 3-D CT findings. High-speed helical CT can scan an object more quickly and clearly than conventional CT, and its biological damage in humans is less than that of other methods.

Differences in response properties of neurons between two delay-tuned areas in the auditory cortex of the mustached bat.

1. The orientation sound (pulse) of the mustached bat, Pteronotus parnellii parnellii, consists of four harmonics (H1-4), each containing a long constant-frequency component (CF1-4) followed by a short frequency-modulated component (FM1-4). The auditory cortex of this species contains several "combination-sensitive" areas: FM-FM, dorsal fringe (DF), ventral fringe (VF), CF/CF, and H1-H2. The FM-FM, DF, and VF areas each consist of neurons tuned to particular delays of echo FMn (n = 2, 3, or 4) from pulse FM1, and have an echo-delay (target-range) axis. This delay axis is from 0.4 to approximately 18 ms in the FM-FM area, to approximately 9 ms in the DF area, and to approximately 5 ms in the VF area. Therefore we hypothesized that the VF area was more specialized for the processing of range information in the terminal phase of echolocation than was the FM-FM area. The aim of our present studies was to find differences in response properties between neurons with best delays shorter than 6 ms in the VF and FM-FM areas and thus to test our hypothesis. 2. In the terminal phase of target-directed flight, the rate of pulse emission becomes higher, pulse duration (in particular, CF duration) becomes shorter, echo delay becomes shorter, and echoes (both the CF and FM components) are less Doppler shifted. Therefore, a "temporal-pattern-simulating (TPS)" stimulus was designed to mimic the train of pulse-echo pairs that would be heard by the bat during the terminal phase, and responses of single neurons to the TPS stimulus and other types of stimuli were recorded from the VF and FM-FM areas of the auditory cortex of unanesthetized bats with a tungsten-wire microelectrode. 3. Best delays of the neurons studied range between 0.9 and 5.5 ms (2.64 +/- 0.72 ms, N = 181) for the VF area, and between 0.6 and 6.0 ms (3.64 +/- 1.14, N = 144) for the FM-FM area. More neurons in the VF area than those in the FM-FM area showed no response or a poor response to the TPS stimulus. Therefore VF neurons are less suited than neurons in the FM-FM area for processing target ranges in the terminal phase of target-directed flight. Facilitative delay-tuning curves were commonly sandwiched between inhibitory delay-tuning curves. The lack of response or poor response to the TPS stimulus can be explained by this inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of combination-sensitive neurons in the ventral fringe area of the auditory cortex of the mustached bat.

1. The orientation sound (pulse) of the mustached bat, Pteronotus parnellii parnellii, consists of long constant-frequency components (CF1-4) and short frequency-modulated components (FM1-4). The auditory cortex of this bat contains several combination-sensitive areas: FM-FM, DF, VA, VF, and CF/CF. The FM-FM area consists of neurons tuned to a combination of the pulse FM1 and the echo FMn (n = 2, 3, or 4) and has an echo-delay (target-range) axis. Our preliminary anatomical studies with tritiated amino acids suggest that the FM-FM area projects to the dorsal fringe (DF) area, which in turn projects to the ventral fringe (VF) area. The aim of our study was to characterize the response properties of VF neurons and to explore the functional organization of the VF area. Acoustic stimuli delivered to the bats were CF tones, FM sounds, and their combinations mimicking the pulse emitted by the mustached bat and the echo. 2. Like the FM-FM and DF areas, the VF area is composed of three types of FM-FM combination-sensitive neurons: FM1-FM2, FM1-FM3, and FM1-FM4. These neurons show little or no response to a pulse alone, echo alone, single CF tone or single FM sound. They do, however, show a strong facilitative response to a pulse-echo pair with a particular echo delay. The essential components in the pulse-echo pair for facilitation are the FM1 of the pulse and the FMn of the echo.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of sudden deafness: carbon dioxide and oxygen inhalation and steroids.

The results of treating 86 patients with sudden deafness are reviewed in this paper. They were treated with intravenous ATP-2Na, Vitamin B1, B6, B12 and C, stellate ganglion block, peroral steroids, cyclandelate and Kallidinogenase. Fifty-one of 86 patients were additionally treated with carbon dioxide and oxygen inhalation. The first 35 patients (no gas inhalation group) and the latter 51 patients (gas inhalation group) were compared with each other concerning hearing improvement, and recovery rate associated with age of the patients, untreated period and effect of steroids. There was no statistical difference between the two groups with regard to these parameters.