Restoration of PPP2CA expression reverses epithelial-to-mesenchymal transition and suppresses prostate tumour growth and metastasis in an orthotopic mouse model.

BACKGROUND: Emergence of castration-resistance in prostate cancer (PCa) is invariably associated with aggressive and metastatic disease. Previously, we reported promotion of castration-resistance upon downregulation of PPP2CA (encoding catalytic subunit of protein phosphatase 2A (PP2A), α-isoform); however, its role in PCa growth and metastasis remained undetermined. METHODS: PPP2CA was overexpressed/silenced in PCa cells by stable transfection. Gene expression was examined by reverse transcription polymerase chain reaction, immunoblot and immunofluorescence analyses, and transcriptional activity measured by luciferase-based promoter-reporter assay. Effect on PCa phenotype was studied in vitro and in orthotopic mouse model, and immunohistochemical/histological analyses performed to assess proliferation/apoptosis and confirm metastatic lesions. RESULTS: An inverse association of PPP2CA expression was observed with epithelial-to-mesenchymal transition (EMT) and aggressive PCa phenotype. PPP2CA restoration resulted in decreased nuclear accumulation and transcriptional activity of ß-catenin/NF-κB, and restitution of their activity abrogated PPP2CA-induced EMT reversal and suppression of PCa invasiveness. Akt mediated PPP2CA loss-induced nuclear accumulation of ß-catenin/NF-κB through inactivation of Gsk3-ß and IκB-α, respectively. Animal studies revealed a suppressive effect of PPP2CA expression on PCa growth and metastasis. CONCLUSIONS: Our findings suggest that PPP2CA downregulation serves as a molecular link between gain of castration-resistance and aggressive PCa phenotype, and its restoration could be an effective preventive/therapeutic approach against the advanced disease.

Increased expression of activation-induced cytidine deaminase is associated with anti-CCP and rheumatoid factor in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID(+) germinal centres were observed in six of the 12 RA synovial samples, and AID(+) cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID(+) cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (P < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10(+)IgM(+)CD20(+) B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (P < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (P

Factors that drive the increasing use of FFPE tissue in basic and translational cancer research.

The decision to use 10% neutral buffered formalin fixed, paraffin embedded (FFPE) archival pathology material may be dictated by the cancer research question or analytical technique, or may be governed by national ethical, legal and social implications (ELSI), biobank, and sample availability and access policy. Biobanked samples of common tumors are likely to be available, but not all samples will be annotated with treatment and outcomes data and this may limit their application. Tumors that are rare or very small exist mostly in FFPE pathology archives. Pathology departments worldwide contain millions of FFPE archival samples, but there are challenges to availability. Pathology departments lack resources for retrieving materials for research or for having pathologists select precise areas in paraffin blocks, a critical quality control step. When samples must be sourced from several pathology departments, different fixation and tissue processing approaches create variability in quality. Researchers must decide what sample quality and quality tolerance fit their specific purpose and whether sample enrichment is required. Recent publications report variable success with techniques modified to examine all common species of molecular targets in FFPE samples. Rigorous quality management may be particularly important in sample preparation for next generation sequencing and for optimizing the quality of extracted proteins for proteomics studies. Unpredictable failures, including unpublished ones, likely are related to pre-analytical factors, unstable molecular targets, biological and clinical sampling factors associated with specific tissue types or suboptimal quality management of pathology archives. Reproducible results depend on adherence to pre-analytical phase standards for molecular in vitro diagnostic analyses for DNA, RNA and in particular, extracted proteins. With continuing adaptations of techniques for application to FFPE, the potential to acquire much larger numbers of FFPE samples and the greater convenience of using FFPE in assays for precision medicine, the choice of material in the future will become increasingly biased toward FFPE samples from pathology archives. Recognition that FFPE samples may harbor greater variation in quality than frozen samples for several reasons, including variations in fixation and tissue processing, requires that FFPE results be validated provided a cohort of frozen tissue samples is available.

Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo.

The promise of gene therapy for health care will not be realized until gene delivery systems are capable of achieving efficient, cell-specific gene delivery in vivo. Here we describe an adenoviral system for achieving cell-specific transgene expression in pulmonary endothelium. The combination of transductional targeting to a pulmonary endothelial marker (angiotensin-converting enzyme, ACE) and an endothelial-specific promoter (for vascular endothelial growth factor receptor type 1, flt-1) resulted in a synergistic, 300,000-fold improvement in the selectivity of transgene expression for lung versus the usual site of vector sequestration, the liver. This combined approach should be useful for the design of other gene delivery systems.

Cell density influences the effect of celecoxib in two carcinoma cell lines.

It has been reported that when ovarian carcinoma cell lines are exposed to various concentrations of celecoxib, a COX-2 inhibitor, cell growth is decreased in a dose dependant manner. To examine further the effect of celecoxib, different cell densities of two carcinoma cell lines were exposed to various concentrations of celecoxib. LNCAP prostate and CAOV3 ovarian carcinoma cells were obtained from the American Type Culture Collection and maintained in Rosewell Park Memorial Institute 1640 and Dulbeceo's modified Eagle's medium, respectively. Each cell line was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotic-antimycotic solution, and placed in a humidified atmosphere containing 5% CO2 at 37 degrees C. After each cell line reached a confluency of 70-80%, 1,000, 2,000, 3,000, 5,000, 7,000 and 10,000 cells/well were seeded in 96 well plates in 100 microl medium/per well for 24 h. Each cell line was exposed to the same concentrations of celecoxib (10-100 microM) at each cell density for 72 h. Cell growth was assessed using a tetrazolium conversion assay. A significant decrease compared to controls was observed in cell growth at each cell density of LNCAP and CAOV3 cells plated with >or=30 microM and >or=50 microM celecoxib, respectively. When the cell growth curves were compared for each cell density at the same concentration of celecoxib, a significant decrease in cell growth was observed when LNCAP cells were plated at 10,000 cells/well and exposed to 10-100 microM celecoxib. At a cell density >or= 5,000 LNCAP cells/well, the inhibitory effect of celecoxib was less. Similarly, a significant decrease in cell growth was observed in CAOV3 cells plated at 1,000 cells/well compared to other cell numbers plated at the same drug concentrations. At a cell density of > 5,000 CAOV3 cells/well, the inhibitory effect of celecoxib was significantly less compared to other cell densities at the same concentration. We observed a more sensitive decrease in cell growth in both carcinoma lines studied at a cell density of 1,000 cells/well with exposure to 10-100 microM celecoxib. Both carcinoma cell lines were less sensitive at a cell density of 5,000 cells/well. Our results suggest that the inhibitory effect of celecoxib may be affected by cell density. Therefore, careful attention must be paid to determining the appropriate cell density for cytotoxicity studies.

Expression of p160erbB-3 and p185erbB-2 in prostatic intraepithelial neoplasia and prostatic adenocarcinoma.

BACKGROUND: Although prostatic adenocarcinoma is the most frequent malignancy among American men, little is known concerning the roles of growth factors, growth factor receptors, or proto-oncogene products in the development and progression of this malignancy. PURPOSE: We examined and compared the expression and cellular distribution of the erbB-3 protein (p160erbB-3) and the erbB-2 protein (p185erbB-2) in various stages of development and progression of prostatic adenocarcinoma. METHODS: Immunoperoxidase staining was used to determine the expression of p160erbB-3 and p185erbB-2 in benign prostatic epithelium, prostatic intraepithelial neoplasia, localized adenocarcinomas (pathologic stages B and C) as well as matching primary and lymph node lesions from patients with stage D adenocarcinoma. In order to test antibody specificity, we used Western blot analysis to examine the expression of p160erbB-3 and p185erbB-2 in selected prostatic adenocarcinomas. RESULTS: Within benign glands, immunostaining for p160erbB-3 or p185erbB-2 was strongest in the basal cells and was typically absent or weak in the luminal (secretory) cells. Expression of both proteins within luminal cells was localized to cell membranes. We examined the expression of p160erbB-3 and p185erbB-2 in the prostatic intraepithelial neoplastic lesions of 22 radical prostatectomy specimens. Like benign glands, the basal cells of these neoplastic lesions typically demonstrated strong to moderate immunostaining when stained with either antibody. In contrast to benign glands, moderate to strong immunostaining for p160erbB-3 and p185erbB-2 was frequently observed within the cytoplasm and cell membranes of the prostatic intraepithelial neoplastic luminal cells. p160erbB-3 and p185erbB-2 were detected within the malignant cells in 28 and 29 of 29 localized adenocarcinomas, respectively. Immunostaining with either antibody was typically moderate to strong in the malignant cells. Both proteins were expressed on the cellular membranes as well as in the cytoplasm of malignant cells. Immunostaining for p160erbB-3 was detected in the matching primary and metastatic lesions (lymph nodes) in 10 of 11 patients with stage D adenocarcinoma. Immunostaining for p185erbB-2 was detected in matching primary and metastatic lesions (lymph nodes) obtained from 16 patients with stage D adenocarcinoma. Western blot analysis confirmed the specificity of the antibodies. CONCLUSIONS: These results suggest that increased expression and changes in the subcellular distribution of both p160erbB-3 and p185erbB-2 represent early events in the development and progression of prostatic adenocarcinomas. The high expression and distribution of both p160erbB-3 and p185erbB-2 are retained both in advanced-stage primary and metastatic tumors.

Midkine promoter-based adenoviral vector gene delivery for pediatric solid tumors.

It is important to develop a system to express therapeutic genes in tumor cells with sufficient selectivity for cancer gene therapy. Midkine (MK) is a newly identified heparin-binding growth factor that is transiently expressed in the early stages of retinoic acid-induced differentiation of embryonal carcinoma cells. It has been reported that many human malignant tumors express high levels of MK mRNA or protein. However, no MK expression is detected in human or mouse liver. These interesting features of MK led us to examine the MK promoter as a candidate for tumor-specific gene expression. We thus developed new recombinant adenoviral (Ad) vectors containing either luciferase reporter gene (AdMKLuc) or herpes simplex thymidine kinase gene (AdMKTK) under the control of the human MK promoter. AdMKLuc achieved relatively high activity in Wilms' tumor (G-401) and neuroblastoma (SK-N-SH) cell lines. In addition, AdMKTK induced marked cell death in response to ganciclovir (GCV) in these same lines. Conversely, very low activity of the MK promoter was observed in mouse liver in vivo compared with the cytomegalovirus promoter. Importantly, AdMKTK + GCV did not induce liver toxicity, whereas substantial toxicity was seen with AdCMVTK + GCV treatment. On the basis of these findings, we conclude that the MK promoter is a candidate tumor-specific promoter for Wilms' tumor or neuroblastoma.

A phase I clinical trial of murine monoclonal antibody D612 in patients with metastatic gastrointestinal cancer.

In a phase I study, 21 patients with metastatic adenocarcinoma of the gastrointestinal tract received the murine monoclonal antibody D612. This antibody is directed at a M(r) 48,000 antigen restrictively expressed on tumors of the gastrointestinal tract and to a limited degree on normal gastrointestinal mucosa. Patients received total doses of 10-180 mg/m2 administered as single or multiple doses of 1-100 mg/m2 over an 8-day period. Dose-limiting toxicity was secretory diarrhea. A single dose of 100 mg/m2 exceeded guidelines for maximal tolerated dose. Higher total doses were achieved in subsequent patients by using repeated administration of lower doses. Three of five patients receiving 60 mg/m2 for 3 doses (180 mg/m2 total dose) experienced grade 3 diarrhea and could not complete the prescribed course. The dose of 40 mg/m2 administered on days 1, 4, and 8 (total dose, 120 mg/m2) has been selected as the dose for phase II studies. The pharmacokinetics of D612 is best described by a one-compartment model with a mean t1/2 of 48 +/- 3 h (SEM). Eighteen of 21 patients developed human anti-mouse antibody (HAMA). Patients who developed high levels of HAMA demonstrated a more rapid clearance of the day 8 dose than those who developed low levels of HAMA. In all patients studied, a component of HAMA was directed at the D612 variable region. With one exception, serum from all patients with detectable antibody to the D612 variable region demonstrated anti-paratope reactivity. Thirty-four % of known metastatic sites demonstrated uptake of radiolabeled D612. There were no objective antitumor responses in this phase I trial. The antitumor effect of D612 in vitro has been shown to be potentiated by interleukin 2 and recombinant human macrophage colony-stimulating factor. A phase II study of D612 administered in combination with cytokines that enhance human effector function is presently ongoing.

Increase of GKLF messenger RNA and protein expression during progression of breast cancer.

Genetic alterations found in carcinomas can alter specific regulatory pathways and provide a selective growth advantage by activation of transforming oncogenes. A subset of these genes, including wild-type alleles of GLI or c-MYC, and activated alleles of RAS or beta-catenin, exhibit transforming activity when expressed in diploid epithelial RK3E cells in vitro. By in vitro transformation of these cells, the zinc finger protein GKLF/KLF-4 was recently identified as a novel oncogene. Although GKLF is normally expressed in superficial, differentiating epithelial cells of the skin, oral mucosa, and gut, expression is consistently up-regulated in dysplastic epithelium and in squamous cell carcinoma of the oral cavity. In the current study, we used in situ hybridization, Northern blot analysis, and immunohistochemistry to detect GKLF at various stages of tumor progression in the breast, prostate, and colon. Overall, expression of GKLF mRNA was detected by in situ hybridization in 21 of 31 cases (68%) of carcinoma of the breast. Low-level expression of GKLF mRNA was observed in morphologically normal (uninvolved) breast epithelium adjacent to tumor cells. Increased expression was observed in neoplastic cells compared with adjacent uninvolved epithelium for 14 of 19 cases examined (74%). Ductal carcinoma in situ exhibited similar expression as invasive carcinoma, suggesting that GKLF is activated prior to invasion through the basement membrane. Expression as determined by Northern blot was increased in most breast tumor cell lines and in immortalized human mammary epithelial cells when these were compared with finite-life span human mammary epithelial cells. Alteration of GKLF expression was confirmed by the use of a novel monoclonal antibody that detected the protein in normal and neoplastic tissues in a distribution consistent with localization of the mRNA. In contrast to most breast tumors, expression of GKLF in tumor cells of colorectal or prostatic carcinomas was reduced or unaltered compared with normal epithelium. The results demonstrate that GKLF expression in epithelial compartments is altered in a tissue-type specific fashion during tumor progression, and suggest that increased expression of GKLF mRNA and protein may contribute to the malignant phenotype of breast tumors.

A standard tissue as a control for histochemical and immunohistochemical staining.

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel(™) and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.

The p44S10 locus, encoding a subunit of the proteasome regulatory particle, is amplified during progression of cutaneous malignant melanoma.

Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.

Assessment of genomic damage in colorectal cancer by DNA fingerprinting: prognostic applications.

PURPOSE: Here we evaluate the prognostic significance of the relative value of genomic damage assessed by DNA fingerprinting in colorectal cancer. MATERIALS AND METHODS: Sixty-three tumor and paired normal mucosa samples were included in the study. Genomic damage was assessed by comparative analysis of paired normal and tumor tissue DNA fingerprints by the arbitrarily primed polymerase chain reaction (AP-PCR). Decreases and increases of intensity in bands were computed and referred to the total number of visualized bands per case. An index reflecting the genomic damage fraction (GDF), with separated values for losses and gains, was obtained for each tumor. This index was used to determine molecular and clinicopathologic correlates after exclusion of eight cases displaying microsatellite instability. RESULTS: Fifty-five cases were considered for the statistical analysis. The average fraction of altered bands per tumor was 0.287+/-0.121. When losses and gains were computed separately, the average fraction of changes was 0.126+/-0.113 and 0.161+/-0.120, respectively. Tumors lacking a ras mutation showed an increased GDF, primarily because of a higher fraction of gains. Tumors that were at advanced Dukes' stages and that were poorly differentiated also displayed a higher GDF. Finally, disease-free survival was significantly diminished in tumors with a GDF greater than 0.314 (P < .001). The prognostic significance of the GDF was independent of Dukes' stage (Cox multivariate analysis, P = .005). CONCLUSION: The degree of genomic damage assessed by unbiased DNA fingerprinting correlates with genotypic, phenotypic, and clinical variables in colorectal carcinoma and may be useful in assessing prognosis in colorectal cancer.

Racial differences in the prognostic usefulness of MUC1 and MUC2 in colorectal adenocarcinomas.

There is a need for new prognostic parameters that could add insights into the aggressiveness of tumors. Because the expression of two well-characterized mucin antigens, MUC1 and MUC2, in colorectal adenocarcinomas (CRCs) has been correlated with the aggressiveness of CRCs, we evaluated the prognostic value of the expression of MUC1 and MUC2 in CRCs collected from African-American and Caucasian patients. Expression of MUC1 and MUC2 was evaluated by immunohistochemistry in 166 archival CRC specimens collected from 58 African-American and 108 Caucasian patients that had been analyzed previously for nuclear accumulation of p53 (p53nac). Univariate Kaplan-Meier and multivariate Cox proportional hazards models were used to determine the prognostic significance of expression of MUC1 and MUC2 in these CRCs. MUC1 expression was more frequent in advanced stage CRCs, whereas MUC2 expression was higher in the mucinous type of CRCs. Although similar proportions of CRCs from African-Americans and Caucasians expressed MUC1 and MUC2, the MUC1 expression was found to be an indicator of high risk of death from CRC in Caucasians (hazard ratio, 2.03; P = 0.038) but not in African-Americans. Furthermore, Caucasians with CRCs exhibiting concomitant expression of MUC1 and p53nac demonstrated the lowest probability of overall survival (log rank test, P = 0.004). No prognostic value was found for MUC2 alone or in combination with p53nac in either group of patients. Expression of MUC1 in CRCs is a valuable indicator of poor prognosis in Caucasian patients. Additionally, combined evaluation of MUC1 and p53nac increases the ability to identify Caucasian patients with aggressive subtypes of CRC and may be useful in selecting or in developing novel therapeutic regimes.

Phase II study of interferon-enhanced 131I-labeled high affinity CC49 monoclonal antibody therapy in patients with metastatic prostate cancer.

Adjuvant Interferon (IFN) was given to increase tumor antigen expression and enhance localization with 131I-labeled CC49 radioimmunotherapy in a Phase II trial for hormone resistant metastatic prostate cancer. Patients received four doses of alpha-IFN (3 x 10(6) IU) s.c. on alternate days, from day -5 to day +1 of 75 mCi/m2 131I-CC49 treatment. Toxicity was well tolerated, with the majority of patients experiencing transient grade 3 or 4 neutropenia and/or thrombocytopenia (maximal at 4-6 weeks). The absorbed dose was >25 Gy in four of eight tumors visualized, which represents an increase of >20 fold over whole body radiation dose. Two patients had radiographic minor responses by 6 weeks post-therapy, whereas five of six patients experiencing pain had symptom relief without radiographic changes. The protocol provided modest antitumor effects (pain relief in five of six patients and two minor radiographic responses). This study suggests that the addition of IFN enhanced tumor uptake and antitumor effects as compared to a prior Phase II trial of 131I-CC49 alone.

Phase II study of dual 131I-labeled monoclonal antibody therapy with interferon in patients with metastatic colorectal cancer.

The combination of COL-1 (anti-CEA) and CC49 (anti-TAG-72) has shown an increase in binding and distribution in colon cancer by immunoperoxidase staining compared to either antibody alone. To overcome tumor heterogeneity and allow delivery of higher radiation dose, 131I-labeled COL-1 and CC49 at a total dose of 75 mCi/m2 (2775 MBq/m2) were simultaneously administered to 14 patients with metastatic colon cancer. alpha-IFN (3 x 10(6) IU) was given s.c. on days -5 to +3 to increase carcinoembryonic antigen and TAG-72 antigen expression. Most patients had mild symptoms during IFN therapy, including mild neutropenia, fever, and malaise, which rapidly subsided after IFN cessation. No acute allergic reactions occurred with radioimmunotherapy; two patients experienced transient, delayed grade 2 arthralgias. Transient neutropenia and/or thrombocytopenia, which was maximal at 4-6 weeks, were consistent side effects without adverse events. All patients had tumor localization, and 13 of 14 patients achieved 4+ (highest grade) localization readings to at least one known site of disease. No objective responses occurred; 4 patients were stable and 10 progressed. Tumor dose estimates varied from 393 to 1327 cGy, including liver and extrahepatic sites. Combining two complementary antibodies and IFN administration appeared to increase localization intensity and radiation doses at tumor sites as compared to historical controls. The amount of radiation delivered to tumor sites was still below that required to cause tumor regressions in metastatic colorectal cancer.

Altered subcellular localization of suppressin, a novel inhibitor of cell-cycle entry, is an independent prognostic factor in colorectal adenocarcinomas.

PURPOSE: Suppressin (SPN), a novel inhibitor of the entry into the cell cycle, has properties of a tumor suppressor gene; however, its role in the development and progression of a human malignancy is not studied. Therefore, we evaluated the status of spn and its prognostic value in human colorectal adenocarcinoma (CRC). EXPERIMENTAL DESIGN: Inhibition of cell proliferation by exogenous/extracellular SPN was assessed by [(3)H]thymidine incorporation. The genetic status of spn in two colon cancer cell lines (LS180 and WiDr) and in a human CRC was determined using direct cDNA sequencing techniques. Phenotypic expression of SPN was evaluated in 105 CRC archival tissues using immunohistochemical methods. Univariate Kaplan-Meier and multivariate Cox proportional hazards models were used to determine the prognostic significance of SPN expression. RESULTS: Exogenous SPN inhibited the proliferation of the LS180 cell line, which also has a mutation in one allele of the spn gene. The spn gene was also mutated in the primary CRC. Expression of SPN was primarily cytoplasmic in nonmucinous CRCs and nuclear in mucinous CRCs. However, the evaluation of 85 nonmucinous CRCs demonstrated that nuclear localization of SPN, nuclear accumulation of p53, and nodal status were independent prognostic indicators with hazard ratios of 2.34, 2.33, and 3.04, respectively. Nuclear localization of SPN plus nuclear accumulation of p53 formed a stronger prognostic indicator (hazard ratios = 5.45) than local nodal status. CONCLUSIONS: This is the first report of genetic alterations in the spn gene in a human malignancy and suggests that genetic alterations in spn and the resulting immunohistochemical phenotypes based on SPN subcellular localization in CRCs may be useful in determining prognosis of patients with subtypes of CRC.

Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and ß-catenin-dependent TCF transcriptional activity.

The cyclic nucleotide phosphodiesterase 10A (PDE10) has been mostly studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we show that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes, as well as in colon tumors from human clinical specimens and intestinal tumors from Apc(Min/+) mice compared with normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 were evident by the ability of small-molecule inhibitors and small interfering RNA knockdown to suppress colon tumor cell growth with reduced sensitivity of normal colonocytes. Stable knockdown of PDE10 by short hairpin RNA also inhibits colony formation and increases doubling time of colon tumor cells. PDE10 inhibition selectively activates cGMP/cGMP-dependent protein kinase signaling to suppress ß-catenin levels and T-cell factor (TCF) transcriptional activity in colon tumor cells. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations suggest a novel role of PDE10 in colon tumorigenesis and that inhibitors may be useful for the treatment or prevention of colorectal cancer.

A novel gene therapy strategy for elimination of prostate carcinoma cells from human bone marrow.

We report a novel means to purge bone marrow of a specific subset of prostate carcinoma cells based on transductional and genetic selectivity. Using both adenovirus-polylysine-DNA complexes and E1A/B-deleted replication-deficient adenoviruses, we have demonstrated a transductional preference of these vectors for the prostate carcinoma cell lines DU 145, LNCaP, and PC-3 over primary human bone marrow cells and the leukemia cell line KG-1. We have also shown a genetic selectivity of an anti-erbB-2 intracellular single-chain antibody (sFv) encoding adenovirus, Ad21, for the erbB-2-positive prostate carcinoma cell lines DU 145 and LNCaP. Delivery of Ad21 resulted in cytotoxicity to the DU 145 and LNCaP, but not PC-3, cell lines and reduced the clonogenic capacity of DU 145 cells cultured alone or mixed with various ratios of irradiated human bone marrow. Finally, quantitative, competitive reverse transcription polymerase chain reaction (QC-RT-PCR) analysis demonstrated that Ad21 could effectively reduce DU 145 and erbB-2-positive primary prostate tumor contamination in bone marrow cultures. Delivery of Ad21 had no effect on the ability of progenitor cells to form colonies. These results suggest that an anti-erbB-2 sFv-encoding adenoviral vector is efficacious for removal of erbB-2-positive prostate carcinoma cells from human bone marrow, and demonstrates a novel method for ex vivo genetic purge of malignant cells from bone marrow for autologous bone marrow transplantation (ABMT) therapy.

Golden Syrian hamsters: a new experimental model for adrenal compensatory hypertrophy.

We report that adrenal compensatory hypertrophy occurs in the golden syrian hamster, an animal secreting cortisol as the primary adrenal glucocorticoid. This response is seen at 3, 6, 10, and 21 days after unilateral adrenalectomy. The response is present in hypophysectomized hamsters and when endogenous ACTH secretion is suppressed by administration of dexamethasone by im injection or by dexamethasone and ACTH using the Alzet osmotic pump. Administration of either aldosterone alone or in combination with dexamethasone and ACTH by Alzet pump completely blocks adrenal compensatory hypertrophy after unilateral adrenalectomy.

Inhibitory and facilitatory areas of the rostral pons mediating ACTH release in the cat.

To define the role of the rostral pons in the control of release of ACTH, we stimulated electrically (30 sec, 200 muA, 50 Hz) 128 sites in the dorsal rostral pons of 20 cats anesthetized with chloralose/urethane. Responses of arterial pressure to electrical stimulation were prevented by lesions placed previously in the medulla. Plasma concentrations of ACTH were measured by radioimmunoassay. Active areas consisted of three regions: 1) lateral inhibitory: Locus subcoeruleus and anteroventral locus coeruleus (mean deltaACTH: -189, -164, -145 pg/ml at 1.5,3.0 and 6.0 min respectively, P less than 0.01);2) intermediate facilitatory:principal locus coeruleus and lateral ventral tegmental nucleus (mean deltaACTH: +81, +68, +37 pg/ml; P less than 0.05); and 3) medial inhibitory: dorsal tegmental nucleus, dorsal raphé and medial ventral tegmental nucleus (mean deltaACTH; -211, -212, -115 pg/ml; P less than 0.01). The former two areas received direct projections from medullary neurons activated or inhibited by atrial stretch, and, in turn, give rise to adrenergic and cholinergic projections to the medial hypothalamus. Since the release of ACTH is inversely correlated with right atrial stretch, the results suggest that the lateral inhibitory area and the intermediate facilitatory area are involved in mediation of changes in release of ACTH in response to hemodynamic changes.