Sp1 and Sp3 activate p21 (WAF1/CIP1) gene transcription in the Caco-2 colon adenocarcinoma cell line.

The CDK inhibitor p21WAF1/CIP1 is a negative regulator of the cell cycle, and its expression is induced during terminal differentiation in vitro and in vivo. Expression of p21 is controlled at the transcriptional level by both p53-dependent and -independent mechanisms. Our previous studies established that p21 is expressed in the Caco-2 adenocarcinoma cell line, and its expression is induced by a p53-independent mechanism during differentiation of these cells. Here we have found that transcription of p21 in Caco-2 cells is controlled primarily by the transcription factors Sp1 and Sp3 through two Sp1 binding sites, Sp1-1 and Sp1-2, located between -119 and -114 bp and between -109 and -104 bp of the p21 promoter, respectively. Sp1 and Sp3 binding to the p21 promoter increased during Caco-2 cell differentiation, while the absolute level of Sp1 did not change and the absolute level of Sp3 increased approximately twofold. Transfection experiments in the SL2 Drosophila cell line that lacks endogenous Sp3 activity demonstrated that Sp1 transactivates the p21 promoter primarily through the Sp1-2 site, while Sp3 acts through the Sp1-1 site. In these cells Sp3 is a stronger transactivator of the p21 promoter than Sp1. Our data suggest that induction of p21 transcription during Caco-2 differentiation is modulated by Sp1/Sp3 interactions with the p21 promoter.

A role for E2F1 in Ras activation of p21(WAF1/CIP1) transcription.

We recently reported that E2F1 could transactivate the p21 promoter via cis-acting elements between -119 to +16 bp of the p21 gene. Here we show that activated V12-H-Ras can induce the p21 promoter through the same region of the p21 promoter by a p53-independent mechanism in NIH3T3 cells. In contrast, activated Ras was not able to induce the p21 promoter in E2F1-/- fibroblasts, suggesting that E2F1 is required for induction of the p21 promoter by activated Ras. Cotransfection of increasing concentrations of dominant negative E2F1 alone, or with dominant negative DP1 into NIH3T3 cells suppressed induction of the p21 promoter by activated Ras. These data suggest that p53-independent induction of the p21 promoter by activated Ras is mediated at least in part by E2F1. Oncogene (2000) 19, 961 - 964.

Energy absorption and propagation in laser-created sparks.

The energy absorption and laser propagation characteristics of air and argon sparks at one atmosphere have been investigated. To create the sparks, 532 nm pulses from a frequency doubled Q-switched Nd : YAG laser are used. We employed 2 ns gated fast photography for studying the time evolution of the kernel at early times. Optical emission spectroscopy is used to infer temperature and density of the sparks. Significant energy absorption by the plasma is observed just above the breakdown threshold. The energy absorption and propagation in the spark indicated that argon plasma is more absorptive than air plasma. The absorption of the spark increases with laser energy, and at higher energies absorption saturation is observed. A spiky behavior is observed in the transmitted temporal profiles of lasers at higher energies and this is explained as due to the formation of a self-regulating regime.

Confinement and dynamics of laser-produced plasma expanding across a transverse magnetic field.

The dynamics and confinement of laser-created plumes expanding across a transverse magnetic field have been investigated. 1.06 microm, 8 ns pulses from a neodymium-doped yttrium aluminum garnet laser were used to create an aluminum plasma which was allowed to expand across a 0.64 T magnetic field. Fast photography, emission spectroscopy, and time of flight spectroscopy were used as diagnostic tools. Changes in plume structure and dynamics, enhanced emission and ionization, and velocity enhancement were observed in the presence of the magnetic field. Photographic studies showed that the plume is not fully stopped and diffuses across the field. The temperature of the plume was found to increase due to Joule heating and adiabatic compression. The time of flight studies showed that all of the species are slowed down significantly. A multiple peak temporal distribution was observed for neutral species.

Flexible CO2 laser system for fundamental research related to an extreme ultraviolet lithography source.

A CO(2) laser system with flexible parameters was developed for fundamental research related to an extreme ultraviolet (EUV) lithography source. The laser is a master oscillator and power amplifier (MOPA) system, consisting of a master oscillator, an externally triggered plasma switch, a preamplifier, a main amplifier, and electronic synchronization units. The laser pulse duration can be varied easily from 10 to 110 ns, with a constant peak power for pulse durations from 25 to 110 ns. The MOPA laser system can also be operated in dual-oscillator mode to produce laser pulse with pulse duration as long as 200 ns and a train of laser pulses with flexible interval. The divergence of the laser beam is 1.3 times the diffraction limit. The laser intensity on the target surface can be up to 8x10(10) W/cm(2). Utilizing this CO(2) MOPA laser system, high conversion efficiency from laser to in-band (2% bandwidth) 13.5 nm EUV emission has been demonstrated over a wide range of laser pulse durations.

Mass-limited Sn target irradiated by dual laser pulses for an extreme ultraviolet lithography source.

A thin Sn film was investigated as a mass-limited target for an extreme ultraviolet (EUV) lithography source. It was found that those energetic ions that are intrinsic with the mass-limited Sn target could be efficiently mitigated by introducing a low-energy prepulse. High in-band conversion efficiency from a laser to 13.5 nm EUV light could be obtained using an Sn film with a thickness down to 30 nm when irradiated by dual laser pulses. It was shown that the combination of dual pulse and inert Ar gas could fully mitigate ions with a low ambient pressure nearly without the penalty of the absorption of the EUV light.

Effect of focal spot size on in-band 13.5 nm extreme ultraviolet emission from laser-produced Sn plasma.

The effect of focal spot size on in-band 13.5 nm extreme ultraviolet (EUV) emission from laser-produced Sn plasmas was investigated for an EUV lithography light source. Almost constant in-band conversion efficiency from laser to 13.5 nm EUV light was noted with focal spot sizes from 60 to 500 microm. This effect may be explained by the opacity of Sn plasmas. Optical interferometry showed that the EUV emission must pass through a longer plasma with higher density when the focal spot is large, and strong reabsorption of EUV light was confirmed by a dip located at 13.5 nm in the spectrum.

Serine proteinase inhibitors produced by human melanoma cell lines.

The human melanoma cell lines M21 and MSM-M2 are shown to produce two similar competitive inhibitors of trypsin, a serine proteinase. These proteinase inhibitors inhibited the serine proteinases trypsin and kallikrein with similar efficiency but did not inhibit plasmin (a serine proteinase) or papain (a thiol proteinase). Active synthesis of the inhibitors during cell culture was indicated by the requirement for cell viability, the increase in inhibitory activity of the supernatant with time, and the incorporation of 35S-methionine into the inhibitors. The two inhibitors were stable to heat (70 degrees C) and extremes of pH. Their molecular weights were estimated at 670 and 250 kD, respectively. A screening of the supernatants of five other human melanoma cell lines by HPLC showed that they all released a similar trypsin inhibitory factor not detected in human or bovine serum. The isolation of these proteinase inhibitors facilitates a study of their putative role in tumor growth.

Endothelin-1 stimulates proliferation of normal airway epithelial cells.

To investigate the influence of endothelins on airway epithelial cell growth, we measured [3H]thymidine incorporation and cell numbers of cultured porcine tracheal epithelial cells in the presence or absence of endothelin-1 or -3 with or without PD-145065 (a combined endothelin-A and -B receptor antagonist), BQ-123 (an antagonist specific for endothelin-A receptors) or phosphoramidon (an inhibitor, in part, of endothelin converting enzymes). We found that endothelin-1 stimulated the proliferation of airway epithelial cells and this response was progressively inhibited by increasing concentrations of either PD-145065 or BQ-123. In contrast to endothelin-1, airway epithelial cells were not responsive to endothelin-3. They also appeared to be producing endothelin-1 endogenously. Phosphoramidon significantly decreased basal growth of cells incubated in the absence of exogenous endothelin, and was associated with a significant diminution in their endothelin production. We conclude that endothelin-1 is mitogenic for porcine airway epithelial cells and may be involved in both autocrine and paracrine control of them.

Inhibitors of lymphocyte activation secreted by human melanoma cell lines.

The concentrated supernatants of nine human melanoma cell line cultures were analyzed for the presence of factors that inhibit in vitro immunological reactions. All cell lines secreted a factor that inhibited LPS-induced proliferation of murine B cells; eight cell lines released a factor that inhibited PHA-induced proliferation of murine T cells; all of the three cell lines investigated secreted a factor that inhibited the allogeneic stimulation of BALB/c spleen cells by mitomycin-C-treated C57BL spleen cells. Further analysis of the M21 cell supernatant indicated that its continuous presence was required for the inhibition of the PHA but not of the LPS response suggesting a different mechanism of action. High levels of PHA, but not of LPS, could overcome the inhibitory effect of M21 supernatants. Fractionation of M21 supernatants by sucrose gradient centrifugation, DEAE chromatography and gel filtration suggested that the anti-PHA and the anti-LPS activities were due to different factors. These factors differed from a serine proteinase inhibitor that is also released by M21 cells. Since these inhibitory factors may have a role in protecting a melanoma tumor from attack by the immune system of the host, their consideration may be helpful in designing protocols for therapy which include methods to boost the antitumor responses of the host.

Interleukin-1 beta increases airway epithelial cell mitogenesis partly by stimulating endothelin-1 production.

To investigate the influence of interleukin-1 beta (IL-1 beta) on airway epithelial cell growth, we measured [3H]thymidine incorporation and cell numbers of cultured porcine tracheal epithelial cells in the presence or absence of human recombinant IL-1 beta with or without the following: goat antiporcine polyclonal antibody to platelet-derived growth factor (PDGF); IL-1 receptor antagonist; indomethacin; PD-145065, a combined endothelin-A and -B receptor antagonist; BQ-123, an antagonist selective for endothelin-A receptors; or phosphoramidon, an inhibitor, in part, of endothelin-converting enzymes, including neutral endopeptidase. We found that IL-1 beta stimulated the proliferation of airway epithelial cells, and this response was inhibited by the IL-1 receptor antagonist and by PD-145065 or BQ-123. However, neither indomethacin nor PDGF antibody was influential. The endothelin receptor antagonists also decreased basal thymidine incorporation by these cells as did phosphormidon, although to a lesser degree. Data from radioimmunoassays indicated that phosphormidon reduced the endogenous production of endothelin-1 from the cells, and IL-1 beta clearly increased it over time. We conclude that IL-1 beta is a stimulant of airway epithelial cell growth, and its mitogenic effects are mediated, in part, by endogenous endothelin-1 production.

Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3.

The cyclin-dependent kinase inhibitor p21((WAF1/CIP1)) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G(1) cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.