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The functions of the retinoblastoma protein (pRb) are in part regulated by reversible and cell cycle-dependent phosphorylation. While the regulation of pRb by cyclin-dependent kinases (Cdks) has been studied extensively, the role(s) of protein phosphatase 1 (PP1) in controlling pRb are only partially understood. In this chapter, we will describe experimental approaches to investigate the interactions between pRb and PP1. Methods will be presented to study the cell cycle-dependent dephosphorylation of pRb by various PP1 isozymes, the specificity of PP1 isozymes for distinct pRb phosphorylation sites, the dephosphorylation of pRb associated with apoptosis, and the cell cycle- and pRb-dependent phosphorylation of PP1.

Studies of families with Birt-Hogg-Dubé syndrome (BHD) have recently revealed protein-truncating mutations in the BHD gene, leading to tumorigenesis of the skin and of different cell types of kidney. To additionally evaluate the role of BHD in kidney tumorigenesis, we studied 39 sporadic renal tumors of different cell types: 7 renal oncocytomas, 9 chromophobe renal cell carcinomas (RCCs), 11 papillary RCCs, and 12 clear cell RCCs. We screened for BHD mutations and identified a novel somatic mutation in exon 13: c.1939_1966delinsT in a papillary RCC. We performed loss of heterozygosity (LOH) analysis on 28 matched normal/tumor sets, of which 10 of 28 (36%) demonstrated LOH: 2 of 6 (33%) chromophobe RCCs, 5 of 6 (83%) papillary RCCs, 3 of 12 (25%) clear cell RCCs, but 0 of 4 renal oncocytomas. BHD promoter methylation status was examined by a methylation-specific PCR assay of all of the tumors. Methylation was detected in 11 of 39 (28%) sporadic renal tumors: 2 of 7 (29%) renal oncocytomas, 1 of 9 (11%) chromophobe RCCs, 4 of 11 (36%) papillary RCCs, and 4 of 12 (33%) clear cell RCCs. Five tumors with methylation also exhibited LOH. Mutation and methylation were absent in 9 kidney cancer cell lines. Our results showed that somatic BHD mutations are rare in sporadic renal tumors. The alternatives, LOH and BHD promoter methylation, are the two possible inactivating mechanisms involved. In conclusion, unlike other hereditary kidney cancer-related genes (i.e., VHL and MET), which are cell type-specific, BHD is involved in the entire spectrum of histological types of renal tumors, suggesting its major role in kidney cancer tumorigenesis.

BACKGROUND: One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity. RESULTS: We have established a cell line for inducing protein expression of the type 1, alpha-isotype, serine/threonine protein phosphatase (PP1alpha). A plasmid encoding a fusion protein of the catalytic subunit of PP1alpha with a 6-histidine peptide (6His) and a peptide from hemagluttinin (HA) was transfected into the UMUC3 transitional cell carcinoma cell line, previously transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo. A stable cell line designated LLWO2F was established by selection with hygromycin B. 6His-HA-PP1alpha protein appeared in cell lysates within two hours following addition of doxycycline to the culture medium. This protein localizes to the nucleus as does endogenous PP1alpha, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1alpha, immunocomplexed 6His-HA-PP1alpha is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels. CONCLUSIONS: These data suggest the existence of an autoregulatory mechanism by which PP1alpha protein levels and activity remain relatively constant. RT-PCR analyses of isolated polysome fractions support the notion that this putative autoregulatory mechanism is exerted, at least in part, at the translational level. Implications of these findings for the study of PP1alpha function in vivo are discussed.

Purpose: The purpose of this study was to compare dental hygiene student radiographic performance using film and charge-coupled device (CCD) systems. Specific objectives were to compare the 1) number of technique errors, 2) error type, 3) retake frequency, and 4) learning experience using both systems., Methods: Twenty-eight subjects exposed two 18-projection full series, one film-based, and one CCD-based system, on a manikin. One examiner, using standardized image quality criteria, evaluated all images. Each subject was assigned to one of four focus groups. Quantitative data were analyzed using ANOVA and Wilcoxon tests. Emic expressions and percentages were used to report qualitative data with themes supported by performance data., Results: A greater number of technique errors occurred using the CCD system than the film system (p < 0.0001), with vertical angulation being the most common error (53%). Using the film-based system, 66% of the errors were due to incorrect horizontal angulation. Subjects exposed an average of 10 retakes per series using the CCD versus 3 retakes using film. Forty-four percent of the subjects felt their most common CCD errors were film placement and vertical angulation compared to 59% who believed horizontal angulation was the main technique error when using film. Difficulty placing the CCD sensor intraorally was expressed by 74%. The CCD system was reported to be beneficial for teaching theory (67%), with 59% preferring film for learning the psychomotor skill., Conclusion: In pre-clinical situations, the CCD system may be an effective tool for teaching technique. Radiographic errors are dependent on the type of image receptor used.

The purpose of this study was to determine if there was a significant difference in the diagnostic quality of the initial full-mouth series (FMS) exposed by first-year dental students after viewing a Computer Assisted Instruction (CAI) module on radiographic technique as compared to those who did not and to determine if students who used the CAI module expressed a preference to using it and would recommend its use prior to exposure. Fifty-nine first-year dental students were randomly selected and assigned to two groups (students who did not view a CAI CD and students who did) before exposing their initial FMS on a Dental X-ray Training and Teaching Replica (DXTTR). For each radiographic series, a radiology faculty member determined the total number of error points based on performance criteria. The Wilcoxon Rank Sum Test was used to determine significance between the two groups, p = .05. After exposing the FMS, students in the second group completed a questionnaire to assess their preference for using the CAI CD. The Sign Rank Test was used to determine preference, p = .05. No significant difference in error points were found to exist between the two groups. Dental students who reviewed the CAI preferred using it and would recommend it to others before exposing their FMS.

Analysis of the viability, differentiation, clonogenicity and function of human stem/progenitor cells requires suitable xenograft models. However, the identification of transplanted cells has been generally difficult. Fluorescence in situ hybridization is a tedious method for analyzing tissues, and localization of transplanted cells with X or Y chromosome probes is limited by the sparse signals produced. Therefore, we examined the possibility of generating either pan-nuclear signals with a total human DNA probe or multiple nuclear signals with a pan-centromeric human DNA probe. The probes were labeled with digoxigenin to make reaction products visible by light microscopy and to allow the use of immunohistochemistry methods incorporating various color schemes to demonstrate specific properties of transplanted cells. The ability to localize all types of nucleated human cells with such probes will facilitate studies of stem cell biology and cell and gene therapy, as well as the development of new animal models.

Tuned Aperture Computed Tomography or TACT is a relatively new technique that transforms correlated two-dimensional images into a three-dimensional display. Associating TACT with digital subtraction radiography (TACT-DSR) may enhance its use in the detection of diseases. This study aims to assess observer performance in the task of detecting simulated bone-gain in periodontal defects, comparing conventional and TACT DSR. Buccal, lingual and proximal pericrestal bony defects in a human dry skull were imaged with and without increments of crushed-bone and wax mixtures using standardized vertical bitewing projections, acquired with an intraoral digital receptor in a tomographic X-ray machine. One hundred and twenty pairs of baseline and follow-up images were obtained for each imaging modality. Post-acquisition processing including image-registration, density-correction and subtraction operation were applied to both transmission images and TACT-slices. Eight calibrated observers evaluated the presence or absence of pericrestal bone-gain using a 5-point confidence scale. Details about the location of periodontal defects and the amount of bone-gain for each subtracted image were recorded and used in the statistical analysis. Observer performance and study design factors were analyzed by means of receiver operating characteristic (ROC) curve analysis and ANOVA. Mean values of areas under ROC curves for bone-gain detection performance were 0.82 for conventional DSR and 0.90 for TACT-DSR. TACT-DSR provided statistically superior detection performance of simulated periodontal bone-gain over conventional DSR for tooth-obscured defects (p = 0.001) and for the smallest bone mass (p = 0.024). The findings support TACT-DSR's potential to provide greater sensitivity and technique flexibility in detecting periodontal bone-gain than standard DSR.

Objective: The objective of this study was to compare the diagnostic efficacy of tuned-aperture computed tomography (TACT) and conventional film for the detection of simulated periodontal defects., Study Design: Periodontal defects were created in 15 premolar-molar interproximal sites selected from 8 cadaver jaw segments. Ten observers viewed 45 image pairs (baseline and follow up) in 4 TACT modalities and film to assess the presence of defect. The TACT modalities were 1st and 2nd generation prototype systems (TACT-1 and TACT-2) with circular (C) and random (R) distribution patterns of source projection (TACT-1C, TACT-1R, TACT-2C, TACT-2R). Observer performance and related factors were analyzed by using receiver operating characteristic and analysis of variance., Results: Mean A(z) values were 0.64 for film, 0.74 for TACT-1C, 0.82 for TACT-1R, 0.64 for TACT-2C, and 0.69 for TACT-2R. TACT-1R was significantly better than film (P < .001), TACT-2C (P < .001) and TACT-2R (P = .007)., Conclusion: TACT-1R provided the best diagnostic performance in the detection of simulated periodontal defects among the 5 modalities compared in this study.