Androgens regulate protein kinase Cdelta transcription and modulate its apoptotic function in prostate cancer cells.

Activation of protein kinase Cdelta (PKCdelta), a member of the novel PKC family, leads to apoptosis in several cell types. Although the molecular bases of PKCdelta activation are being unfolded, limited information is available on the mechanisms that control its expression. Here, we report that in prostate cancer cells PKCdelta is tightly regulated by androgens at the transcriptional level. Steroid depletion from the culture medium causes a pronounced down-regulation of PKCdelta protein and mRNA in androgen-sensitive LNCaP prostate cancer cells, an effect that is rescued by the androgen R1881 in an androgen receptor (AR)-dependent manner. Analysis of the PKCdelta promoter revealed a putative androgen responsive element (ARE) located 4.7 kb upstream from the transcription start site. Luciferase reporter assays show that this element is highly responsive to androgens, and mutations in key nucleotides in the AR-binding consensus abolish reporter activity. Furthermore, using chromatin immunoprecipitation assays, we determined that the AR binds in vivo to the PKCdelta ARE in response to androgen stimulation. Functional studies revealed that, notably, androgens modulate phorbol 12-myristate 13-acetate (PMA)-induced apoptosis in LNCaP cells, an effect that is dependent on PKCdelta. Indeed, androgen depletion or AR RNA interference severely impaired the apoptotic function of PKCdelta or the activation of p38, a downstream effector of PKCdelta in LNCaP cells--effects that can be rescued by restoring PKCdelta levels using an adenoviral delivery approach. Our studies identified a novel hormonal mechanism for the control of PKCdelta expression via transcriptional regulation that fine-tunes the magnitude of PKCdelta apoptotic responses.

Protein kinase C and prostate carcinogenesis: targeting the cell cycle and apoptotic mechanisms.

A series of both genetic and epigenetic factors have been implicated in the genesis and progression of prostate cancer. Recent evidence revealed that protein kinase C (PKC) isozymes play a crucial role in the control of cell proliferation and apoptosis in prostate cancer models, as well as in the transition from an androgen-dependent to an androgen-independent status. Indeed, PKCalpha and PKCdelta promote apoptosis in androgen-dependent prostate cancer cells. Due to the relevance of PKC isozymes in the control of cell cycle, both in G1/S and G2/M, the elucidation of such complex intracellular networks using cellular and animal models has become of outmost importance. In this review, we present the current knowledge on the regulation of apoptosis and tumorigenicity by PKC isozymes and the functional roles of cell cycle regulators in prostate carcinogenesis. The development of animal models where overexpression of discrete PKCs or cell cycle regulators is targeted to the prostate will greatly contribute to the understanding of the molecular basis of the disease, and more importantly, it will have profound implications for the development of novel strategies for prostate cancer therapy.

Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway.

Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKC alpha and the novel PKC delta isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574-7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645-655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKC alpha in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells and suggest that both p38 MAPK and Akt play critical roles as downstream effectors of PKC isozymes in this cellular model.

Na(+)/I(-) symporter activity requires a small and uncharged amino acid residue at position 395.

Active iodide uptake in the thyroid is mediated by the Na(+)/I(-) symporter (NIS), a key plasma membrane glycoprotein. Several NIS mutations have been shown to cause I(-) transport defect, a condition that, if untreated, can lead to congenital hypothyroidism and, ultimately, cretinism. The study of I(-) transport defect-causing NIS mutations provides valuable insights into the structure-function and mechanistic properties of NIS. Here we report the thorough analysis of the G395R NIS mutation. We observed no I(-) uptake activity at saturating or even supersaturating external I(-) concentrations in COS-7 cells transiently transfected with G395R NIS cDNA, even though we demonstrated normal expression of G395R NIS and proper targeting to the plasma membrane. Several amino acid substitutions at position 395 showed that the presence of an uncharged amino acid residue with a small side chain at position 395 is required for NIS function, suggesting that glycine 395 is located in a tightly packed region of NIS. Substitutions of large amino acid residues at position 395 resulted in lower V(max) without affecting K(m) values for I(-) and Na(+), suggesting that these residues hamper the Na(+)/I(-) coupling reaction.

Segmentation of suspicious clustered microcalcifications in mammograms.

We have developed a multistage computer-aided diagnosis (CAD) scheme for the automated segmentation of suspicious microcalcification clusters in digital mammograms. The scheme consisted of three main processing steps. First, the breast region was segmented and its high-frequency content was enhanced using unsharp masking. In the second step, individual microcalcifications were segmented using local histogram analysis on overlapping subimages. For this step, eight histogram features were extracted for each subimage and were used as input to a fuzzy rule-based classifier that identified subimages containing microcalcifications and assigned the appropriate thresholds to segment any microcalcifications within them. The final step clustered the segmented microcalcifications and extracted the following features for each cluster: the number of microcalcifications, the average distance between microcalcifications, and the average number of times pixels in the cluster were segmented in the second step. Fuzzy logic rules incorporating the cluster features were designed to remove nonsuspicious clusters, defined as those with typically benign characteristics. A database of 98 images, with 48 images containing one or more microcalcification clusters, provided training and testing sets to optimize the parameters and evaluate the CAD scheme, respectively. The results showed a true positive rate of 93.2% and an average of 0.73 false positive clusters per image. A comparison of our results with other reported segmentation results on the same database showed comparable sensitivity and at the same time an improved false positive rate. The performance of the CAD scheme is encouraging for its use as an automatic tool for efficient and accurate diagnosis of breast cancer.

Simulation model of mammographic calcifications based on the American College of Radiology Breast Imaging Reporting and Data System, or BIRADS.

RATIONALE AND OBJECTIVES: The authors developed and evaluated a method for the simulation of calcification clusters based on the guidelines of the Breast Imaging Reporting and Data System of the American College of Radiology. They aimed to reproduce accurately the relative and absolute size, shape, location, number, and intensity of real calcifications associated with both benign and malignant disease. MATERIALS AND METHODS: Thirty calcification clusters were simulated by using the proposed model and were superimposed on real, negative mammograms digitized at 30 microns and 16 bits per pixel. The accuracy of the simulation was evaluated by three radiologists in a blinded study. RESULTS: No statistically significant difference was observed in the observers' evaluation of the simulated clusters and the real clusters. The observers' classification of the cluster types seemed to be a good approximation of the intended types from the simulation design. CONCLUSION: This model can provide simulated calcification clusters with well-defined morphologic, distributional, and contrast characteristics for a variety of applications in digital mammography.

Interpretation of calcifications in screen/film, digitized, and wavelet-enhanced monitor-displayed mammograms: a receiver operating characteristic study.

RATIONALE AND OBJECTIVES: The acceptance of filmless digital mammography is currently limited by digitization and display drawbacks, as well as bias toward hard-copy interpretation. In the current study, we evaluated a wavelet-based image enhancement method for the filmless interpretation of breast calcifications. METHODS: A set of 100 mammograms (58 with calcification clusters) was digitized at 105 microns and 4,096 gray levels per pixel and was processed with nonlinear filters and wavelets. Standard receiver operating characteristic analysis was performed by four radiologists, who independently read the films, the unprocessed digital images, and unprocessed and wavelet-enhanced digital images presented simultaneously. RESULTS: Statistical differences were observed between screen/film and unprocessed digitized mammography displayed on monitors. Differences were not significant when wavelet enhancement was included in the monitor display. Interobserver variation in the digitized reading was greater than in film reading, but the wavelet enhancement reduced the difference. CONCLUSION: Wavelet-enhanced digital mammograms may assist radiologists in diagnosing calcifications directly from computer monitors and may compensate for current technologic limitations. A study with a larger data-base is needed before this method is accepted for clinical use.