Spontaneous hypertension ion cross-suckled rats.

Genetically normotensive Sprague-Dawley rats nursed by spontaneously hypertensive foster mothers develop sustained high blood pressure. Some factor related to the genetically programmed hypertension of the foster mother is probably transmitted to the infant rats through her milk. Alternatively, the hyperkinetic behavior of the mother may activate a hypertensinogen in young having the proper constellation of genes.

PGF2alpha-associated vascular smooth muscle hypertrophy is ROS dependent and involves the activation of mTOR, p70S6k, and PTEN.

Prostaglandin F2alpha (PGF2alpha) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2alpha-induced VSMC hypertrophy we examined the ability of the PGF2alpha analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3beta (GSK-3beta), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3beta, PTEN and ERK1/2 but not JNK phosphorylation. Whereas inhibition of phosphatidylinositol 3-kinase (PI3K) by LY-294002 blocked fluprostenol-induced changes in total protein content, pre-treatment with rapamycin or with the MEK1/2 inhibitor U0126 did not. Taken together, these findings suggest that fluprostenol-induced changes in A7r5 hypertrophy involve mTOR translocation and occur through PI3K-dependent mechanisms.

Matrix Metalloproteinases -14, -9 and -2 are Localized to the Podosome and Involved in Podosome Development in the A7r5 Smooth Muscle Cell.

AIM: The purpose of the study was to localize matrix metalloproteinase (MMP)-14, -9, and -2 in the A7r5 smooth muscle cell and to understand the interaction between these MMPs and the cytoskeleton. This interaction was observed under non-stimulating and phorbol 12, 13-dibutyrate (PDBu)-stimulating conditions. METHODS: Confocal microscopy was utilized to define the localizations of MMPs and tissue inhibitor of matrix metalloproteinases (TIMPs) in the A7r5 cell and to determine interaction between MMPs and the cytoskeleton. Under PDBu-stimulating conditions, the presence of MMP active forms and activity by gel zymography was evaluated in the A7r5 cell. Actin and microtubule-polymerization inhibitors were used to evaluate MMP interaction with the cytoskeleton and the cytoskeleton was observed on matrix and within a Type I collagen gel. RESULTS: MMP-14, -9, and -2 were localized to the podosome in the A7r5 smooth muscle cell and interactions were seen with these MMPs and the actin cytoskeleton. PDBu-stimulation induced increases in the protein abundance of the active forms of the MMPs and MMP-2 activity was increased. MMPs also interact with a-actin and not ß-tubulin in the A7r5 cell. Galardin, also known as GM-6001, was shown to inhibit podosome formation and prevented MMP localization to the podosome. This broad spectrum MMP inhibitor also prevented collagen gel contraction and prevented cell adhesion and spreading of A7r5 cells within this collagen matrix. CONCLUSION: MMPs are important in the formation and function of podosomes in the A7r5 smooth muscle cell. MMPs interact with a-actin and not ß-tubulin in the A7r5 cell. Podosomes play an important role in cell migration and understanding the function of podosomes can lead to insights into cancer metastasis and cardiovascular disease.

Binding of human IgG to cultured urogenital tumor cells.

Binding of human IgG by the Fc portion of the immunoglobulin molecule was detected on established human tumor cell lines by indirect immunofluorescence microscopy, cytofluorography, quantitative absorption, and rosette formation with the use of antibody-coated erythrocytes. Of the nonlymphoid tumors tested, IgG binding was restricted to the cell membranes of certain prostate and urinary bladder tumor cell lines. Although most cell lines tested shared a common antigenic determinant with monocytes and granulocytes, these cells did not express T- and B-cell antigens, the complement 3b receptor, or bind a monoclonal antibody specific for the Fc receptor expressed on human neutrophils. The facts that IgG binding was present on long-term established tumor lines and was not influenced by in vitro passage provide evidence that these properties are intrinsic to the tumor cells and may play some role in the pathophysiology of these tumors.

Disulfide bonding of a human prostate tumor-associated membrane antigen recognized by monoclonal antibody D83.21.

Previous reports from our laboratory have described the binding specificity of a monoclonal antibody, D83.21, prepared by fusing P3X63/Ag8 mouse myeloma cells with mouse lymphocytes immunized against the DU145 human prostate adenocarcinoma cell line. The D83.21 monoclonal antibody displayed preferential binding to human prostate and bladder carcinoma cell lines and tissues. This antibody was not reactive with a variety of other normal and malignant human cell lines or tissues. Immunofluorescence analysis indicated that the D83.21 antigen was located on the plasma membrane. Biochemical characterization of the target antigen was performed by subjecting detergent-soluble extracts of [3H]glucosamine-labeled cells to D83.21 monoclonal antibody affinity chromatography. The radioactive material that was specifically bound and eluted from the affinity column was analyzed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis and fluorography. In the absence of 2-mercaptoethanol, the antigen displayed two prominent bands with molecular weights of 180 and 110 kilodaltons. In the reduced form, the antigen is composed of an Mr 60,000 heavy chain and an Mr 28,000 light chain. The antigen was further resolved using two-dimensional (intact/reduced) sodium dodecyl sulfate: polyacrylamide gel electrophoresis. These analyses indicated that the Mr 180,000 chain was composed entirely of Mr 60,000 subunits, whereas the Mr 110,000 band consisted only of Mr 28,000 subunits. The antigen recognized by the D83.21 monoclonal antibody is therefore a membrane glycoprotein with a subunit structure cross-linked together through disulfide bonds.

Comparative study of monoclonal antibodies TURP-27 and HNK-1: their relationship to neural cell adhesion molecules and prostate tumor-associated antigens.

The murine monoclonal antibodies TURP-27 and HNK-1 have been shown to detect antigens which are heavily expressed by benign prostatic hyperplasia and carcinoma of the prostate. Western blot analysis of prostate extracts showed that monoclonal antibodies TURP-27 and HNK-1 bound glycoproteins with molecular weights of 180,000, 140,000, 120,000, 100,000, 90,000, and 69,000. Studies have shown that the HNK-1 carbohydrate epitope may be involved in cell adhesion and that it is a component of several characterized adhesion proteins. TURP-27 was found to bind at least three of these adhesion proteins: neural cell adhesion molecules; myelin-associated glycoprotein; and a second myelin glycoprotein, P0. Western blot analysis of prostate extracts showed that an antineural cell adhesion molecule serum bound the Mr 180,000 and 140,000 proteins. Based on reciprocal blocking and chemical tests, it was determined that the TURP-27 and HNK-1 epitopes are not identical. These data imply that the TURP-27 epitope may be a variant of the HNK-1 epitope or that the two epitopes are closely linked and that the TURP-27 and HNK-1 epitopes on prostate cells are positioned on neural cell adhesion molecule-like proteins.

Monoclonal antibody PD41 recognizes an antigen restricted to prostate adenocarcinomas.

A monoclonal antibody (MAb) designated PD41 (IgG1k) was generated by hyperimmunizing BALB/c mice with a membrane preparation prepared from a moderately to poorly differentiated prostate carcinoma surgical specimen. The immunohistochemical reactivity of MAb PD41 was shown to be highly restricted to the ductal epithelia and secretions of prostate adenocarcinoma tissues. Sixty-five % of the prostate tumor specimens were stained with MAb PD41, whereas no staining of the fetal or benign prostate specimens was observed. PD41 reacted minimally with normal prostate tissues, with less than 1% of the epithelial cells staining. This MAb did not react with nonprostate carcinomas or to a variety of normal human tissues. Using both radioimmunoassay and immunofluorescent procedures, several cultured human tumor cell lines, human blood cells, and purified antigens to prostate-specific antigen and prostatic acid phosphatase also were found not to express the PD41 antigen. MAb PD41 also was shown to bind to the target antigen present in seminal plasma obtained from prostate carcinoma patients but not to seminal plasma from normal donors. Immunoblots of gel-separated components of prostate carcinoma tissue extracts indicate that the molecular weight of the proteins carrying the PD41 antigenic determinant can differ among individual tumors, ranging from Mr 90,000 to greater than 400,000. However, in seminal plasma from prostate cancer patients, the predominant component recognized by PD41 is the diffuse Mr greater than 400,000 band. It appears that this monoclonal antibody may recognize a prostate carcinoma-associated mucin-like antigen, which is preferentially expressed on prostate carcinomas, and therefore, may be a useful marker to distinguish benign prostate hyperplasia from prostate carcinoma.

Cloning and characterization of UROC28, a novel gene overexpressed in prostate, breast, and bladder cancers.

A novel gene, designated UROC28, was identified by an agarose gel-based differential display technique, and it was found to be up-regulated in prostate, breast, and bladder cancer. Expression of UROC28 was also up-regulated in prostate cancer cells in the presence of androgens as demonstrated by relative quantitative reverse transcription-PCR. The elevated expression of this gene was observed to increase in surgically removed tissues concomitantly with rising Gleason grade and was most elevated in metastatic tissue. UROC28 protein was detected in serum by Western slot blot analyses, and a significant higher UROC28 protein level was found in sera of prostate cancer individuals compared with normal individuals and individuals with nonmalignant prostatic hyperplasia. Northern analyses in normal tissues showed that the UROC28 cDNA hybridizes to two mRNAs at about 2.1 and 2.5 kb. Nucleic acid sequence analyses indicated that these two alternatively spliced mRNA variants differ only at the 3' untranslated region. These two mRNAs encode the same protein with 135 amino acids. Bioinformation analyses suggest that there is a possible transmembrane domain from amino acid aa34 to aa50, three protein kinase-C phosphorylation sites at aa62 (SQK), aa89 (TMK), and aa94 (SMK), and one myristylation site at aa118 (GLECCL). Genomic Southern hybridization and chromosomal mapping demonstrated that UROC28 is encoded by a single copy of gene at chromosome 6q23-24. In situ hybridization and immunohistochemistry experiments further confirmed up-regulation of this gene in prostate and breast cancers with the expression localizing to the glandular epithelium. This gene did not demonstrate increased expression in lung and colon cancer tissues.

Flt3-ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer.

We assessed the in vivo efficacy of Flt3-ligand (Flt3-L) treatment in C57BL/6 mice bearing a well-established MHC class I-negative prostate carcinoma TRAMP-C1. Flt3-L immunotherapy was initiated approximately 30 days after tumor inoculation, a time when > or =80% of the mice had palpable TRAMP-C1 tumors. Treatment with Flt3-L at 10 microg/day for 21 consecutive days suppressed TRAMP-C1 tumor growth and induced tumor stabilization (P = 0.0337). Enhanced tumor regression was demonstrated at a higher dose of 30 microg/day (P < 0.0001). Tumors excised from mice treated with Flt3-L were smaller than carrier-treated controls and contained a more pronounced mixed inflammatory cell infiltrate primarily composed of mphi. In regressor nice, tumors reappeared at the site of injection when Flt3-L therapy was terminated. When the experiment was repeated with MHC class I-positive TRAMP-C1 cells, tumor stabilization and/or regression was again observed after treatment (P < 0.0001); however, once again, tumors reappeared after the termination of therapy despite an extended treatment schedule (35 days). MHC class I-negative variants were present in tumors isolated from carrier- and Flt3-L-treated mice, and this phenotype could be reversed by IFN-gamma treatment in vitro. Thus, Flt3-L treatment of mice with preexisting transplantable prostate tumors results in tumor regression that is dose-dependent and accompanied by a pronounced mixed-cell inflammatory tumor infiltrate. However, disease relapse was invariably observed after the termination of therapy, which suggests that Flt3-L treatment of advanced MHC- prostate cancers will require adjuvant modalities to achieve a durable response.

Immunohistochemical localization of prostate carcinoma-associated antigens.

The immunoperoxidase technique was used to study the localization and distribution of antigens reactive with two monoclonal antibodies, D83.21 and P6.2, produced against cultured prostate tumor cells, in formalin-fixed, paraffin-embedded histological sections of human tissues. Monoclonal D83.21 reacted with 11 of 19 (58%) primary prostate carcinomas and 1 of 6 (17%) metastatic tumors, whereas monoclonal P6.2 reacted with 14 of 19 (68%) primary and 4 of 6 (67%) metastatic prostate tumors. Neither antibody reacted with five primary prostate tumors and one metastatic prostate tumor. In some tumor cells, the antigens recognized by these monoclonals were localized in either the cytoplasm or cell membrane, while in other tumor cells, both diffuse cytoplasmic and membrane or focal staining patterns were observed. In addition to the variable staining patterns, antigenic heterogeneity was also noted within most prostate tumors examined. Two types of staining variability were observed: (a) tumor cells in one area of the tissue section stained positive, but in another area they did not react with the antibody; and (b) both stained and unstained tumor cells were adjacent to each other. These results would suggest that a panel of monoclonals will be required to detect the different subpopulations of prostate tumor cells. Neither antibody reacted with 6 normal or 12 benign prostate tissues, nor any of a variety of other normal human tissues except for staining of the proximal tubules of normal kidneys. The antigen detected by P6.2 demonstrated a wider tissue distribution being found on bladder, breast, lung, and pancreatic tumors, whereas the antigen recognized by D83.21 was restricted to prostate and bladder carcinomas. These antibodies may have clinical applicability for the identification of prostate tumor cells in biopsy specimens and for immunohistopathological classification of prostate carcinomas.

Monoclonal antibodies to human prostate and bladder tumor-associated antigens.

Monoclonal antibodies to human prostate adenocarcinoma membrane antigens were produced by fusion of P3X63/Ag8 mouse myeloma cells with spleen cells from BALB/c mice immunized against the prostate cancer cell line DU145. The hybrids were screened for antibody production using glutaraldehyde-fixed cells in a solid-phase radioimmunoassay. Antibody-binding specificity was also checked by quantitative adsorption, membrane immunofluorescence, and complement-dependent cytotoxicity assays. A hybridoma clone (83.21) was isolated that secreted antibodies which preferentially bound to several prostate and bladder cancer cell lines but did not bind to a variety of other normal and malignant human cell lines. This antibody also reacted with a cytomegalovirus-transformed human embryonic lung cell line but not to normal human embryonic lung cells. Quantitative adsorption studies demonstrated that the 83.21 monoclonal antibody was strongly reactive to membrane preparations from human prostate adenocarcinoma tissue and a liver metastasis of prostate carcinoma. Little or no binding activity was observed against two other prostate carcinomas, bening prostatic hyperplasia, normal prostate, or normal liver. Binding studies indicate that the 83.21 monoclonal antibody does not bind to alpha-fetoprotein, carcinoembryonic antigen, prostatic acid phosphatase, human leukocyte antigen, beta 2-microglobulin, HLA-Dr antigens, fibronectin, or prostate antigen. The data indicate that we have isolated a monoclonal antibody that binds to an antigen(s) expressed by several urogenital carcinoma cell lines as well as human prostate tumor tissue and that the antibody is not directed against well-known human tumor cell markers.

Quantitation of serum prostate-specific membrane antigen by a novel protein biochip immunoassay discriminates benign from malignant prostate disease.

The lack of a sensitive immunoassay for quantitating serum prostate-specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic/prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. PSMA was captured from serum by anti-PSMA antibody bound to ProteinChip arrays, the captured PSMA detected by surface-enhanced laser desorption/ionization mass spectrometry, and quantitated by comparing the mass signal integrals to a standard curve established using purified recombinant PSMA. The average serum PSMA value for prostate cancer (623.1 ng/ml) was significantly different (P < 0.001) from that for benign prostate hyperplasia (117.1 ng/ml) and the normal groups (age <50, 272.9 ng/ml; age >50, 359.4 ng/ml). These initial results suggest that serum PSMA may be a more effective biomarker than prostate-specific antigen for differentiating benign from malignant prostate disease and warrants additional evaluation of the surface-enhanced laser desorption/ionization PSMA immunoassay to determine its diagnostic utility.

Human prostate tissue antigens defined by murine monoclonal antibodies.

BALB/c mice were hyperimmunized against a membrane preparation derived from a pool of transurethral resection specimens which included three benign prostatic hyperplasia and one prostate adenocarcinoma tissue samples. The activated lymphocytes were fused with the NS-1 mouse myeloma cell line, and supernatants from immunogen-reactive hybridomas were screened for antibody binding activity using a solid-phase radioimmunoassay against the Calu-1 human lung adenocarcinoma cell line and several membrane preparations derived from various normal human tissues. Hybridoma cultures secreting antibodies which did not appear cross-reactive were doubly cloned by limiting dilution and screened against a large panel of membrane preparations derived from normal prostate, benign prostatic hyperplasia, and prostate adenocarcinoma tissues as well as samples obtained from a variety of normal human tissues. The monoclonal antibodies were also evaluated against 24 normal, virally transformed, and malignant human cell lines. Two monoclonal antibodies were isolated which demonstrated a restricted binding activity to prostate antigens and were not widely cross-reactive with nonprostate normal tissues or cell lines. These antibodies were designated TURP-27 (IgG3, k) and TURP-73 (IgG2a, k). Both of these monoclonal antibodies were reactive against formalin-fixed, paraffin-embedded tissues in the immunoperoxidase assay and were subsequently tested against a variety of normal, hyperplastic, and malignant human tissues. These studies indicated that TURP-27 may be directed against a new prostate organ-associated marker and that TURP-73 is directed against an antigen expressed on prostate and a limited number of other tissues.

Aging alters vascular mechanotransduction: pressure-induced regulation of p70S6k in the rat aorta.

Physical forces are important regulators of vascular structure and function though it is unknown how aging may affect the ability of the vasculature to respond to mechanical stimuli. We investigated the pressure-induced activation of ribosomal S6-kinase (p70S6k) and its pathway-related proteins (Akt, GSK-3beta, SHP-2, PTEN) in aortae from young adult (6 month), aged (30 month), and very aged (36 month) Fischer 344 x Brown Norway F1 hybrid rats. With aging, the aortic tissue content of Akt. SHP-2, and PTEN was significantly increased while total p70S6k and GSK-3beta were unchanged. By comparison, the basal phosphorylation of p70S6k at Thr 389 and Thr 421/Ser 424 was increased ( approximately 40%) and unchanged, respectively, while Akt decreased (approximately 37%), GSK-3beta was unchanged, SHP-2 increased (approximately 73.5%), and PTEN increased (approximately 120%) in the aortae of very aged rats. Acute pressurization of aortae resulted in similar increases in phosphorylation of Akt among the different age groups. By comparison, pressure-induced phosphorylation of p70S6k at Thr 389, GSK-3beta and SHP-2 decreased; whereas, PTEN dephosphorylation was increased in 36-month versus 6-month aortae. The results indicate marked alterations in the p70S6k signaling pathway with aging. The implications of these findings on age-associated vessel remodeling are discussed.

Prostate-specific membrane antigen levels in sera from healthy men and patients with benign prostate hyperplasia or prostate cancer.

Prostate-specific membrane antigen (PSMA) serum levels have been proposed to be of prognostic significance in patients with advanced prostate disease. The objective of the present study was to confirm PSMA serum expression by Western blot techniques, to determine whether such data could assist in the differentiation of benign from malignant prostatic disease, and to determine the suitability of serum PSMA measurements in predicting recurrent or progressive prostate malignancies. We measured PSMA, a transmembrane glycoprotein identified in prostate epithelial cells, in the sera of 236 normal individuals and cancer patients by Western blot analysis. Within the normal male population, PSMA levels increase with age and were found to be significantly elevated in subjects more than 50 years of age when compared to those of younger men. We did not confirm previous reports that serum PSMA measurements could distinguish late-stage prostate carcinoma from early-stage prostate carcinoma, nor did we find PSMA to be more effective than prostate-specific antigen in monitoring prostate cancer patient prognosis. Furthermore, we found elevated serum PSMA in healthy females, and, similar to the healthy male population, the levels increased with age, with the highest levels found in the sera from breast cancer patients. These latter observations further support that PSMA is not a specific biomarker for prostate cancer and that a variety of normal and diseased tissue may contribute to the serum levels of PSMA.

An endogenous 'hypertensive factor' enhances the voltage-dependent calcium current.

The effects of an immunoaffinity-purified putative endogenous hypertensive factor (HF) on voltage-dependent calcium current in frog cardiac myocytes were assessed. In 9 out of 10 cells, HF reversibly increased the peak amplitude of the calcium current. HF increased peak calcium current density at -5 mV from a control level of 1.8 +/- 1.3 pA/pF (mean +/- SD) to 4.4 +/- 2.0 pA/pF. HF shifted the peak of the calcium current-voltage relationship in the hyperpolarizing direction. HF shifted the voltage dependence of the inactivation of the calcium current to more negative potentials with prepulses from -80 to 0 mV, but the inactivation was not affected with prepulses more positive than 0 mV. Modulation of the voltage-dependent calcium current by HF may be the mechanism underlying its pressor effects.

Overexpression of p53 in prostate carcinoma is associated with improved overall survival but not predictive of response to radiotherapy.

p53 gene mutations are among the most common specific genetic alterations in human cancer. Inactivation of p53 and subsequent protein accumulation has been implicated in a variety of human malignancies and associated with prostate cancer progression. In this study, we assessed p53 protein overexpression and gene mutations in prostate carcinoma and investigated associations between p53 alterations and clinicopathological parameters, survival, and response to radiotherapy. We evaluated 58 archival formalin-fixed, paraffin-embedded prostate carcinomas to detect abnormal p53 nuclear protein accumulation using immunohistochemistry. p53 mutational status of tumor DNA was evaluated using polymerase chain reaction-single-strand conformation polymorphism analysis of exons 5-9 and confirmed by direct DNA sequencing. Univariate and multivariate statistical analysis was used to determine the association of p53 status with clinical characteristics and response to radiotherapy. Overexpression of p53 was detected in 42 (72%) of 58 primary prostate carcinomas, but was undetectable in 7 samples of benign prostatic hyperplasias or 5 samples of normal prostate tissue. p53 exon 5-9 mutations were detected in 8 (14%) of 58 patient specimens. p53 mutational status, but not overexpression, was associated with higher Gleason scores (p=0.0145). Neither p53 overexpression nor mutation was associated with clinical stage, biochemical disease-free probability, or predictive of response to radiotherapy. p53 protein accumulation was inversely associated with improved overall survival (p=0.0108). Our studies demonstrate that p53 protein accumulation is a frequent alteration in prostate cancer. The disparity between p53 protein overexpression and p53 exon 5-9 mutations suggests the possibility of mutations outside this region or stabilization of wild-type p53 by alternative mechanisms. In our patient population, p53 protein overexpression or mutational status was not predictive of outcome in patients treated with radiation therapy. Additional studies are needed to further evaluate the association between p53 protein overexpression and improved overall survival.

Proteomic strategies for biomarker identification: progress and challenges.

The simplest definition of a biomarker is a molecule that indicates an alteration in physiology from normal. A more practical definition of a biomarker would require clinical utility of this molecule. In this sense, the biomarker would specifically and sensitively reflect a disease state and could be used for diagnosis as well as for disease monitoring during and following therapy. The need for such biomarkers in all clinical fields is urgent, since the current arsenal of biomarkers is sadly deficient and, in most cases, non-specific. In this review, we discuss strategies that use a proteomics approach to identify novel biomarkers and give examples of recent studies employing these strategies.

Proteomic approaches within the NCI early detection research network for the discovery and identification of cancer biomarkers.

In the postgenome era, proteomics provides a powerful approach for the analysis of normal and transformed cell functions, for the identification of disease-specific targets, and for uncovering novel endpoints for the evaluation of chemoprevention agents and drug toxicity. Unfortunately, the genomic information that has greatly expounded the genetic basis of cancer does not allow an accurate prediction of what is actually occurring at the protein level within a given cell type at any given time. The gene expression program of a given cell is affected by numerous factors in the in vivo environment resulting from tissue complexity and organ system orchestration, with cells acting in concert with each other and responding to changes in their microenvironment. Repositories of genomic information can be considered master "inventory lists" of genes and their maps, which need to be supplemented with protein-derived information. The National Cancer Institute's Early Detection Research Network is employing proteomics, or "protein walking", in the discovery and evaluation of biomarkers for cancer detection and for the identification of high-risk subjects. Armed with microdissection techniques, including the use of Laser Capture Microdissection (LCM) to procure pure populations of cells directly from human tissue, the Network is facilitating the development of technologies that can overcome the problem of tissue heterogeneity and address the need to identify markers in easily accessible biological fluids. Proteomic approaches complement plasma-based assays of circulating DNA for cancer detection and risk assessment. LCM, coupled with downstream proteomics applications, such as two-dimensional polyacrylamide gel electrophoresis and SELDI (surface enhanced laser desorption ionization) separation followed by mass spectrometry (MS) analysis, may greatly facilitate the characterization and identification of protein expression changes that track normal and disease phenotypes. We highlight recent work from Network investigators to demonstrate the potential of proteomics to identify proteins present in cancer tissues and body fluids that are relevant for cancer screening.

Evidence for dramatically increased bone turnover in spontaneously hypertensive rats.

Using the 3H-tetracycline model, whole-body skeletal bone resorption was compared among male and female spontaneously hypertensive (SHR) rats and normotensive Wistar Kyoto (WKy) and Sprague-Dawley (SD) rats. Immature animals undergoing rapid skeletal growth and bone sculpting showed a tendency for decreased indices of skeletal resorption in females compared with males. By 24 weeks of age, the indices of the rate of resorption and extent of metabolically reactive bone in male rats were decreased a mean of 68% and 74%, respectively, compared with values obtained at 8 weeks. By comparison, values for 24-week-old females decreased only 26% and 56%, respectively, evidence for a significantly elevated level of resorptive activity in mature females compared with males in each of the 3 rat strains. Within-sex comparisons of 24-week-old animals indicated that bone resorptive activity was similar between normotensive male and normotensive female groups. By comparison, the resorptive activity was significantly increased in both male and female hypertensive rats compared with normotensive controls. This condition was exaggerated in female hypertensive rats, which showed an approximate 81% and 44% increase in the indices of rate of resorption and extent of metabolically reactive bone compared with normotensive WKy controls. The results indicate a marked sexual dichotomy in the decline of skeletal bone resorptive activity following maturation and slowing of skeletal growth. They further indicate a significant elevation of whole skeleton bone turnover in male SHR rats and dramatically increased bone turnover in female SHR rats.