Cell cycle and biochemical effects of PD 0183812. A potent inhibitor of the cyclin D-dependent kinases CDK4 and CDK6.

Progression through the G1 phase of the cell cycle requires phosphorylation of the retinoblastoma gene product (pRb) by the cyclin D-dependent kinases CDK4 and CDK6, whose activity can specifically be blocked by the CDK inhibitor p16(INK4A). Misregulation of the pRb/cyclin D/p16(INK4A) pathway is one of the most common events in human cancer and has lead to the suggestion that inhibition of cyclin D-dependent kinase activity may have therapeutic value as an anticancer treatment. Through screening of a chemical library, we initially identified the [2,3-d]pyridopyrimidines as inhibitors of CDK4. Chemical modification resulted in the identification of PD 0183812 as a potent and highly selective inhibitor of both CDK4 and CDK6 kinase activity, which is competitive with ATP. Flow cytometry experiments showed that of the cell lines tested, only those expressing pRb demonstrated a G1 arrest when treated with PD 0183812. This arrest correlated in terms of incubation time and potency with a loss of pRb phosphorylation and a block in proliferation, which was reversible. These results suggest a potential use of this chemical class of compounds as therapeutic agents in the treatment of tumors with functional pRb, possessing cell cycle aberrations in other members of the pRb/cyclin D/p16(INK4A) pathway.

Fourier analysis of corneal topography data after photorefractive keratectomy.

PURPOSE: To evaluate the relation between measures of corneal power and refractive error after photorefractive keratectomy (PRK) by applying fast Fourier transform (FFT) to computerized corneal topography data. SETTING: Corneal Diseases and Excimer Laser Clinic, Sunderland Eye Infirmary, Sunderland, England, and University of Dundee Department of Ophthalmology, Dundee, Scotland. METHODS: Twenty-six left eyes of consecutive patients treated by PRK with a VISX Twenty-Twenty excimer laser were retrospectively analyzed. Preoperative and 3, 6, and 12 month postoperative data were studied. Changes in corneal parameters derived from corneal topography data using the FFT were compared with changes in refractive status vectors (spherical equivalent and astigmatic cosine and sine values). RESULTS: Although highly correlated (r2 = 0.8839), the change in FFT-derived corneal spherical equivalent underestimated the change in refractive spherical equivalent by 25.5% over the 12 month follow-up. Decentration, measured by the 1-cycle FFT harmonic, increased significantly from a mean preoperative value of 0.12 mm +/- 0.07 (SD) to 0.51 +/- 0.35 mm 12 months postoperatively. CONCLUSIONS: The FFT is a powerful method for extracting clinically meaningful descriptors from corneal topography data; however, care must be taken when interpreting refractive changes from corneal data.

Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor.

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.

Biochemical and antiproliferative properties of 4-[ar(alk)ylamino]pyridopyrimidines, a new chemical class of potent and specific epidermal growth factor receptor tyrosine kinase inhibitor.

The tyrosine kinase inhibitors PD 69896, 153717, and 158780, which belong to the chemical class 4-[ar(alk)ylamino]pyridopyrimidines, have been characterized with respect to enzymology, target specificity, and antiproliferative effects in tumor cells. These compounds were competitive inhibitors with respect to ATP against purified epidermal growth factor (EGF) receptor tyrosine kinase and inhibited EGF receptor autophosphorylation in A431 human epidermoid carcinoma with IC50 values of 2085, 110, and 13 nM, respectively. Onset of inhibition was immediate once cells were exposed to these compounds, whereas recovery of receptor autophosphorylation activity after the cells were washed free of the compound was dependent on inhibitory potency. Thus, full activity returned immediately after removal of PD 69896 but required 8 hr after exposure to PD 158780. PD 158780 was highly specific for the EGF receptor in Swiss 3T3 fibroblasts, inhibiting EGF-dependent receptor autophosphorylation and thymidine incorporation at low nanomolar concentrations while requiring micromolar levels for platelet-derived growth factor- and basic fibroblast growth factor-dependent processes. PD 158780 inhibited heregulin-stimulated phosphorylation in the SK-BR-3 and MDA-MB-453 breast carcinomas with IC50 values of 49 and 52 nM, respectively, suggesting that the compound was active against other members of the EGF receptor family. The antiproliferative effects of this series of compounds against A431 cells correlated precisely with the inhibitory potency against EGF receptor autophosphorylation. PD 158780 reduced clone formation in soft agar of fibroblasts transformed by EGF, EGF receptor, or the neu oncogene but not ras or raf, further demonstrating its high degree of specificity. Finally, this compound was active against clone formation in several breast tumors having different expression patterns of the erbB family, indicating an anticancer utility in tumors expressing these receptors.

Computer simulation of centration effects on corneal-topography analysis of excimer laser photorefractive keratectomy ablations.

Excimer laser photorefractive keratectomy (PRK) procedures photoablate the central optical zone, potentially altering the corneal vertex normal and thus the corneal topography (CT) system reference axis. Anterior corneal surface coordinates (x, y, and z) were computer generated to simulate spherical and PRK-ablated corneal surfaces. Power maps were reconstructed by using surface tangents relative to various assumed CT reference axes, the original and displaced vertex normals. The simulation process eliminated focus, alignment, and surface-reconstruction errors. A simulated 5.00-D myopic PRK decentered 1 mm from the vertex produced a new corneal vertex 0.13 mm from the original position. Axial power-map generation by using this new reference axis produced a spherical keratectomy with astigmatic untreated periphery. This computer simulation demonstrates that reference axis changes are significant for even modest PRK decentration and can explain apparent steepening in untreated corneal areas. CT users should be aware that pre- and postoperative CT power maps may not be directly comparable.

Corneal topography bow-tie pattern: artifact of videokeratoscopy?

PURPOSE: To test the hypothesis that the bow-tie corneal topography pattern results from corneal asphericity in the presence of astigmatism. METHODS: Astigmatic color-coded power maps using different shape factors were computer generated. Each simulation was based on the calculation of dioptric power at 20 points along each of 180 hemimeridia, for a total of 3600 points. The calculations were made independent of the capture or measurement of video-keratographs. These simulations were compared to power maps taken from clinical records. RESULTS: A shape factor of 1.00 resulted in a spherocylinder color-coded map with straight-edged sectors of power. The familiar bow-tie pattern was generated using an elliptical model with a shape factor of less than 1.00. This pattern was reversed by modeling the cornea as an oblate ellipsoid using a shape factor greater than 1.00. CONCLUSIONS: By simple alteration of the amount of corneal asphericity through manipulation of the shape factor, computer simulation showed that this surface characteristic is responsible for the bow-tie pattern observed in corneal topography power maps.

Amino-terminal sequence determinants for substrate recognition by platelet-derived growth factor receptor tyrosine kinase.

The specificity of protein kinases has been shown to be influenced by residues near the phosphoaccepting amino acid. To examine the determinants for platelet-derived growth factor receptor (PDGFR) tyrosine kinase specificity, a peptide library with three degenerate positions N-terminal to tyrosine was constructed. After reaction with PDGFR, the most abundant phosphopeptides were isolated by immunoaffinity chromatography on a column containing monoclonal anti-phosphotyrosine antibody. Further separation of bound phosphopeptides with reverse-phase HPLC led to the identification of three optimal substrates for PDGFR: Ala-Ala-Asn-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, Ala-Ala-Asn-Arg-Thr-Tyr-Ala-Ala-Arg-Arg-Gly and Ala-Ala-Leu-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, where underlined residues are in the degenerate positions of the peptide library. Kinetic analyses of the three individual peptides (synthesized separately) showed these peptides to be among the best reported substrates for PDGFR. Our results expand the range of amino acid residues that have been shown to serve as recognition elements for receptor tyrosine kinases.

Expression, purification and characterization of focal adhesion kinase using a baculovirus system.

Focal adhesion kinase (FAK) has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus was generated which contains the mouse FAK cDNA cloned into a histidine tag transfer vector. Synthesis of the immunoreactive recombinant protein (baculovirus focal adhesion kinase (BFAK) Mr approximately 125,000) in infected Sf9 cells was detected 23 h postinfection, with maximal accumulation occurring at 48 h postinfection. BFAK constituted 5.4% of total soluble protein in the insect cell lysate and represented 19 mg/liter culture (approximately 2 x 10(9) cells). The enzyme was active as a protein tyrosine kinase in both SF9 cells and in vitro. Purification to near homogeneity was achieved by nickel chelation chromatography. A yield of 5 mg of purified active BFAK was consistently produced from 1 liter of infected insect cells. BFAK tyrosine kinase activity was characterized physically using poly(Glu-Tyr) as a substrate. BFAK activity required the presence of a divalent cation and exhibited a preference for Mn2+ over Mg2+. Maximal tyrosine kinase activity was attained at pH 7.2. Steady-state kinetic analysis with respect to ATP concentration did not conform to simple Michaelis-Menten kinetics and exhibited a Hill coefficient of much less than 1. Km values for ATP using native and autophosphorylated BFAK were 6.7 +/- 1.0 and 4.3 +/- 0.2 microM, respectively. Kcat values were 13.9 +/- 1.9 and 8.9 +/- 0.3 nmol/min/mg BFAK. Steady-state kinetics with respect to the peptide substrate did fit the Michaelis-Menten equation and exhibited a Km value of 2.4 +/- 0.3 micro/ml.

The relation between corneal and total astigmatism.

Using computer-assisted videokeratoscopy we measured corneal astigmatism and compared these results over a range of corneal zone diameters with total ocular astigmatism derived by subjective refraction. Videokeratoscopes permit a more detailed analysis of the power distribution within a given corneal surface area, enabling comparison to the total astigmatism for equivalent aperture sizes. Although there were significant individual variations, the group average data supports the traditional view of a linear relation between corneal and total astigmatism. This was true across the range of apertures tested from 2 to 7 mm, with the coordinates of the relation being consistent with that of the modified Javal's rule; namely a slope of 1 and an intercept of approximately 0.50 D against-the-rule residual astigmatism.

Diethylene glycol mono butyl ether concentrations in room air from application of cleaner formulations to hard surfaces.

Diethylene glycol monobutyl ether (DGBE) is a solvent used in some liquid hard surface cleaners. We evaluated the inhalation component of consumer exposure in the home to DGBE from the use of cleaning products containing up to 9% DGBE. Several experiments were conducted with restricted room air flow, exaggerated amounts of cleaning solutions, and no rinsing in order to develop an exposure scenario that would exceed exposures likely encountered by consumers. DGBE vapors in the air were monitored by collection on charcoal tubes, followed by desorption and quantitation by gas chromatography. Air was collected from the centre of the room and from the breathing zone of the person doing the washing task. Room air concentrations of DGBE showed peak values between one and three hours after task initiation; DGBE concentrations then gradually decreased with time. Peak concentrations did not exceed 1.6 ppmv. The total DGBE in the air at the time of maximum air concentrations accounted for only 1 to 3% of the DGBE on the washed surfaces. The person doing the washing task was exposed to average DGBE concentrations in the breathing zone below 0.8 ppmv in all experiments. The methods described for measuring DGBE concentrations in air are generally applicable to other solvents and easily adaptable to various experimental situations.

Binding of a pancreatic nuclear protein is correlated with amylase enhancer activity.

The mouse amylase gene Amy-2.2 is expressed at high levels specifically in the acinar cells of the pancreas. The region between -172 and -110 of this gene includes sequence elements common to pancreas-specific genes. Nuclear proteins with specific affinity for this region were partially purified from rat pancreas. The consensus element of another pancreas-specific gene, elastase 1, competes for protein binding to the amylase sequences. Binding was localized by DNase I protection to the sequence -156 to -122. Site-directed mutagenesis of this sequence resulted in concomitant loss of protein binding and enhancer activity. Photo-affinity labelling of pancreatic nuclear extracts identified one predominant binding protein with a molecular weight of approximately 75 kDa. The data indicate that binding of this nuclear protein is essential for the enhancer activity of this pancreas-specific element.

Glucocorticoid and developmental regulation of amylase mRNAs in mouse liver cells.

We characterized alpha-amylase expression in the hepatoma cell line Hepa 1-6 and in normal mouse liver. Both Amy-1 and Amy-2 were expressed in Hepa 1-6 and were regulated by glucocorticoids. Transcription in the hepatoma cells was initiated at the same start sites as in mouse tissues. Glucocorticoid treatment increased the abundance of Amy-1 and Amy-2 transcripts by 10 to 20-fold. This increase was detected within 4 h and was maximal by 24 h. The pattern of amylase expression in this hepatoma cell line accurately reflects amylase expression in the liver in vivo. During liver development, we observed a large increase in the abundance of Amy-1 transcripts just before birth, at a time when circulating glucocorticoids are also elevated. Adult mouse liver expressed Amy-1 and Amy-2 at levels comparable to those of fully induced hepatoma cells. Liver is thus a likely source of both amylase isozymes in mouse serum. These studies demonstrate that Amy-2 expression is not limited to the pancreas but also occurs at a low level in liver cells.