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Introduction: Bladder cancer is a heterogenous disease and the emerging knowledge on molecular classification of bladder tumors may impact treatment decisions based on molecular subtype. Pre-clinical models representing each subtype are needed to test novel therapies. Carcinogen-induced bladder cancer models represent heterogeneous, immune-competent, pre-clinical testing options with many features found in the human disease., Methods: Invasive bladder tumors were induced in C57BL/6 mice when continuously exposed to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in the drinking water. Tumors were excised and serially passed by subcutaneous implantation into sex-matched syngeneic C57BL/6 hosts. Eight lines were named BBN-induced Urothelium Roswell Park (BURP) tumor lines. BURP lines were characterized by applying consensus molecular classification to RNA expression, histopathology, and immune profiles by CIBERSORT. Two lines were further characterized for cisplatin response., Results: Eight BURP tumor lines were established with 3 male and 3 female BURP tumor lines, having the basal/squamous (BaSq) molecular phenotype and morphology. BURP-16SR was established from a male mouse and has a stromal-rich (SR) molecular phenotype and a sarcomatoid carcinoma morphology. BURP-19NE was established from a male mouse and has a neuroendocrine (NE)-like molecular phenotype and poorly differentiated morphology. The established BURP tumor lines have unique immune profiles with fewer immune infiltrates compared to their originating BBN-induced tumors. The immune profiles of the BURP tumor lines capture some of the features observed in the molecular classifications of human bladder cancer. BURP-16SR growth was inhibited by cisplatin treatment, while BURP-24BaSq did not respond to cisplatin., Discussion: The BURP lines represent several molecular classifications, including basal/squamous, stroma-rich, and NE-like. The stroma-rich (BURP-16SR) and NE-like (BURP-19NE) represent unique immunocompetent models that can be used to test novel treatments in these less common bladder cancer subtypes. Six basal/squamous tumor lines were established from both male and female mice. Overall, the BURP tumor lines have less heterogeneity than the carcinogen-induced tumors and can be used to evaluate treatment response without the confounding mixed response often observed in heterogeneous tumors. Additionally, basal/squamous tumor lines were established and maintained in both male and female mice, thereby allowing these tumor lines to be used to compare differential treatment responses between sexes., Competing Interests: SS is currently an employee of GSK and holds stock/ownership interests, However, all work and analysis presented in the paper was done during her time at Roswell Park. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shah, Gillard, Wrobel, Karasik, Moser, Mastri, Long, Sule, Brackett, Huss and Foster.)

The evolving paradigm of the molecular classification of bladder cancer requires models that represent the classifications with less heterogeneity. Robust transcriptome based molecular classifications are essential to address tumor heterogeneity. Patient derived models (PDMs) are a powerful preclinical tool to study specific tumor compartments. We tested if the consensus molecular subtype analysis was applicable to PDMs and evaluated the tumor compartment each model represents. PDMs derived from surgical specimens were established as xenografts (PDX), organoids (PDO), and spheroids (PDS). The surgical specimens and PDMs were molecularly characterized by RNA sequencing. PDMs that were established in immune deficient mice or in vitro significantly downregulated transcripts related to the immune and stromal compartments compared to the surgical specimens. However, PDMs upregulate a patient-specific bladder cancer cell signal which allowed for analysis of cancer cell pathways independent of the tumor microenvironment. Based on transcriptomic signatures, PDMs are more similar to their surgical specimen than the model type; indicating that the PDMs retained unique features of the tumor from which the PDM was derived. When comparing models, PDX models were the most similar to the surgical specimen, while PDO and PDS models were most similar to each other. When the consensus molecular subtype classification system was applied to both the surgical samples and the three PDMs, good concordance was found between all samples indicating that this system of classification can be applied to PDO and PDS models. PDMs reduce tumor heterogeneity and allow analysis of tumor cells while maintaining the gene expression profile representative of the original tumor., Competing Interests: None., (AJCEU Copyright © 2021.)

The mechanistic target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation and survival in response to the availability of energy sources and growth factors. Cancer development and progression is often associated with constitutive activation of the mTOR pathway, thus justifying mTOR inhibition as a promising approach to cancer treatment and prevention. However, development of previous rapamycin analogues has been complicated by their induction of adverse side effects and variable efficacy. Since mTOR pathway regulation involves multiple feedback mechanisms that may be differentially activated depending on the degree of mTOR inhibition, we investigated whether rapamycin dosing could be adjusted to achieve chemopreventive efficacy without side effects. Thus, we tested the efficacy of two doses of a novel, highly bioavailable nanoformulation of rapamycin, Rapatar, in a mouse prostate cancer model (male mice with prostate epithelium-specific Pten -knockout). We found that the highest efficacy was achieved by the lowest dose of Rapatar used in the study. While both doses tested were equally effective in suppressing proliferation of prostate epithelial cells, higher dose resulted in activation of feedback circuits that reduced the drug's tumor preventive efficacy. These results demonstrate that low doses of highly bioavailable mTOR inhibitor, Rapatar, may provide safe and effective cancer prevention., Competing Interests: CONFLICTS OF INTEREST M.P.A. and A.V.G. served as consultants for Everon Biosciences, Inc.; O.V.C. and A.V.G. are co-founders and shareholders of Everon Biosciences, Inc.

Toll-like receptor 5 (TLR5) is considered an attractive target for anticancer immunotherapy. TLR5 agonists, bacterial flagellin and engineered flagellin derivatives, have been shown to have potent antitumor and metastasis-suppressive effects in multiple animal models and to be safe in both animals and humans. Anticancer efficacy of TLR5 agonists stems from TLR5-dependent activation of nuclear factor-κB (NF-κB) that mediates innate and adaptive antitumor immune responses. To extend application of TLR5-targeted anticancer immunotherapy to tumors that do not naturally express TLR5, we created an adenovirus-based vector for intratumor delivery, named Mobilan that drives expression of self-activating TLR5 signaling cassette comprising of human TLR5 and a secreted derivative of Salmonella flagellin structurally analogous to a clinical stage TLR5 agonist, entolimod. Co-expression of TLR5 receptor and agonist in Mobilan-infected cells established an autocrine/paracrine TLR5 signaling loop resulting in constitutive activation of NF-κB both in vitro and in vivo. Injection of Mobilan into primary tumors of the prostate cancer-prone transgenic adenocarcinoma of the mouse prostate (TRAMP) mice resulted in a strong induction of multiple genes involved in inflammatory responses and mobilization of innate immune cells into the tumors including neutrophils and NK cells and suppressed tumor progression. Intratumoral injection of Mobilan into subcutaneously growing syngeneic prostate tumors in immunocompetent hosts improved animal survival after surgical resection of the tumors, by suppression of tumor metastasis. In addition, vaccination of mice with irradiated Mobilan-transduced prostate tumor cells protected mice against subsequent tumor challenge. These results provide proof-of-concept for Mobilan as a tool for antitumor vaccination that directs TLR5-mediated immune response toward cancer cells and does not require identification of tumor antigens.

Background: Lysine-specific demethylase 1A (LSD1) is a key regulator of the androgen (AR) and estrogen receptors (ER), and LSD1 levels correlate with tumor aggressiveness. Here, we demonstrate that LSD1 regulates vitamin D receptor (VDR) activity and is a mediator of 1,25(OH) 2 -D 3 (vitamin D) action in prostate cancer (PCa)., Methods: Athymic nude mice were xenografted with CWR22 cells and monitored weekly after testosterone pellet removal. Expression of LSD1 and VDR (IHC) were correlated with tumor growth using log-rank test. TRAMP tumors and prostates from wild-type (WT) mice were used to evaluate VDR and LSD1 expression via IHC and western blotting. The presence of VDR and LSD1 in the same transcriptional complex was evaluated via immunoprecipitation (IP) using nuclear cell lysate. The effect of LSD1 and 1,25(OH) 2 -D 3 on cell viability was evaluated in C4-2 and BC1A cells via trypan blue exclusion. The role of LSD1 in VDR-mediated gene transcription was evaluated for Cdkn1a , E2f1 , Cyp24a1 , and S100g via qRT-PCR-TaqMan and via chromatin immunoprecipitation assay. Methylation of Cdkn1a TSS was measured via bisulfite sequencing, and methylation of a panel of cancer-related genes was quantified using methyl arrays. The Cancer Genome Atlas data were retrieved to identify genes whose status correlates with LSD1 and DNA methyltransferase 1 (DNMT1). Results were correlated with patients' survival data from two separate cohorts of primary and metastatic PCa., Results: LSD1 and VDR protein levels are elevated in PCa tumors and correlate with faster tumor growth in xenograft mouse models. Knockdown of LSD1 reduces PCa cell viability, and gene expression data suggest a dual coregulatory role of LSD1 for VDR, acting as a coactivator and corepressor in a locus-specific manner. LSD1 modulates VDR-dependent transcription by mediating the recruitment of VDR and DNMT1 at the TSS of VDR-targeted genes and modulates the epigenetic status of transcribed genes by altering H3K4me2 and H3K9Ac and DNA methylation. Lastly, LSD1 and DNMT1 belong to a genome-wide signature whose expression correlates with shorter progression-free survival and overall survival in primary and metastatic patients' samples, respectively., Conclusions: Results demonstrate that LSD1 has a dual coregulatory role as corepressor and coactivator for VDR and defines a genomic signature whose targeting might have clinical relevance for PCa patients.

The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (calcitriol) has antiproliferative effects in non-aggressive prostate cancer, however, its effects in more aggressive model systems are still unclear. In these studies, effects of calcitriol and a less-calcemic vitamin D analog, QW-1624F2-2 (QW), were tested in vivo, using the aggressive autochthonous transgenic adenocarcinoma of mouse prostate (TRAMP) model. To study prevention of androgen-stimulated prostate cancer, vehicle, calcitriol (20 µg/kg), or QW (50 µg/kg) were administered to 4 week-old TRAMP mice intraperitoneal (i.p.) 3×/week on a MWF schedule for 14 weeks. Calcitriol and QW slowed progression of prostate cancer as indicated by reduced urogenital tract (p = 0.0022, calcitriol; p = 0.0009, QW) and prostate weights (p = 0.0178, calcitriol; p = 0.0086, QW). However, only calcitriol increased expression of the pro-differentiation marker, cadherin 1 (p = 0.0086), and reduced tumor proliferation (p = 0.0467). By contrast, neither vitamin D analog had any effect on castration resistant prostate cancer in mice treated pre- or post-castration. Interestingly, although vitamin D showed inhibitory activity against primary tumors in hormone-intact mice, distant organ metastases seemed to be enhanced following treatment (p = 0.0823). Therefore, TRAMP mice were treated long-term with calcitriol to further examine effects on metastasis. Calcitriol significantly increased the number of distant organ metastases when mice were treated from 4 weeks-of-age until development of palpable tumors (20-25 weeks-of-age)(p = 0.0003). Overall, data suggest that early intervention with vitamin D in TRAMP slowed androgen-stimulated tumor progression, but prolonged treatment resulted in development of a resistant and more aggressive disease associated with increased distant organ metastasis.

Rationale: Couples share distress as well as potential personal growth (PG) after a cancer diagnosis. It is essential for professionals to learn more about the ways couples cope together with adversity. Dyadic results may help to understand controversial results in the PG literature and inform clinicians in optimizing psychological support for couples., Objective: We examine the temporal and dyadic development of PG among patients and their intimate partners. In addition, life threat is examined as a potential factor influencing PG after cancer diagnosis., Methods: We assessed PG using the Personal Growth Inventory in a clinically representative, mixed-type and mixed-stage cancer cohort (N = 154 couples) 6 and 12 months after cancer diagnosis. Medical data on cancer diagnoses and treatments were collected from physicians. Actor-partner interdependence models were applied., Results: PG was reported by patients and their partners. Women (either as patients or partners) reported more PG than male patients or partners. PG remained relatively stable over 6 months and was related to whether the patient was receiving curative or palliative treatment. Female patients experienced less PG 6 months after the cancer diagnosis and if treatment was curative. Male patients experienced less PG if their partners experienced PG, and the treatment was curative., Conclusions: Dyadic growth is a phenomenon not limited to breast cancer or female patient couples and may represent a form of dyadic coping. Patients and partners seem to develop individual and dyadic growth, depending on a combination of gender and life threat. Psycho-oncology services may want to promote both couple level coping and support in order to optimize cancer care.

Objective: Assessment and treatment of psychological distress in cancer patients was recognized as a major challenge. The role of spouses, caregivers, and significant others became of salient importance not only because of their supportive functions but also in respect to their own burden. The purpose of this study was to assess the amount of distress in a mixed sample of cancer patients and their partners and to explore the dyadic interdependence., Methods: An initial sample of 154 dyads was recruited, and distress questionnaires (Hospital Anxiety and Depression Scale, Symptom Checklist 9-Item Short Version and 12-Item Short Form Health Survey) were assessed over four time points. Linear mixed models and actor-partner interdependence models were applied., Results: A significant proportion of patients and their partners (up to 40%) reported high levels of anxiety, depression, psychological distress, and low quality of life over the course of the investigation. Mixed model analyses revealed that higher risks for clinical relevant anxiety and depression in couples exist for female patients and especially for female partners. Although psychological strain decreased over time, the risk for elevated distress in female partners remained. Modeling patient-partner interdependence over time stratified by patients' gender revealed specific effects: a moderate correlation between distress in patients and partners, and a transmission of distress from male patients to their female partners., Conclusions: Our findings provide empirical support for gender-specific transmission of distress in dyads coping with cancer. This should be considered as an important starting point for planning systemic psycho-oncological interventions and conceptualizing further research., (Copyright © 2013 John Wiley & Sons, Ltd.)

Understanding the biology of Waldenström macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secretes human immunoglobulin M (h-IgM) with κ-light chain restriction identical to the primary tumor. The cell line has a modal chromosomal number of 46 and harbors chromosomal changes such as deletion of 6q21, monoallelic deletion of 9p21 (CDKN2A), 13q14 (RB1) and 18q21 (BCL-2), with a consistent amplification of 14q32 (immunoglobulin heavy chain; IgH) identical to its founding tumor sample. The clonal relationship is confirmed by identical CDR3 length and single nucleotide polymorphisms as well as a matching IgH sequence of the cell line and founding tumor. Both also harbor a heterozygous, non-synonymous mutation at amino acid 265 in the MYD88 gene (L265P). The cell line expresses most of the cell surface markers present on the parent cells. Overall, RPCI-WM1 represents a valuable model to study Waldenström macroglobulinemia.

Advanced prostate cancer commonly metastasizes to bone, but transit of malignant cells across the bone marrow endothelium (BMEC) remains a poorly understood step in metastasis. Prostate cancer cells roll on E-selectin(+) BMEC through E-selectin ligand-binding interactions under shear flow, and prostate cancer cells exhibit firm adhesion to BMEC via β1, β4, and αVβ3 integrins in static assays. However, whether these discrete prostate cancer cell-BMEC adhesive contacts culminate in cooperative, step-wise transendothelial migration into bone is not known. Here, we describe how metastatic prostate cancer cells breach BMEC monolayers in a step-wise fashion under physiologic hemodynamic flow. Prostate cancer cells tethered and rolled on BMEC and then firmly adhered to and traversed BMEC via sequential dependence on E-selectin ligands and β1 and αVβ3 integrins. Expression analysis in human metastatic prostate cancer tissue revealed that β1 was markedly upregulated compared with expression of other β subunits. Prostate cancer cell breaching was regulated by Rac1 and Rap1 GTPases and, notably, did not require exogenous chemokines as β1, αVβ3, Rac1, and Rap1 were constitutively active. In homing studies, prostate cancer cell trafficking to murine femurs was dependent on E-selectin ligand, β1 integrin, and Rac1. Moreover, eliminating E-selectin ligand-synthesizing α1,3 fucosyltransferases in transgenic adenoma of mouse prostate mice dramatically reduced prostate cancer incidence. These results unify the requirement for E-selectin ligands, α1,3 fucosyltransferases, β1 and αVβ3 integrins, and Rac/Rap1 GTPases in mediating prostate cancer cell homing and entry into bone and offer new insight into the role of α1,3 fucosylation in prostate cancer development.

Chronic inflammation leads to the formation of a pro-tumorigenic microenvironment that can promote tumor development, growth and differentiation through augmentation of tumor angiogenesis. Prostate cancer (CaP) risk and prognosis are adversely correlated with a number of inflammatory and angiogenic mediators, including Toll-like receptors (TLRs), NF-κB and vascular endothelial growth factor (VEGF). Peroxiredoxin 1 (Prx1) was recently identified as an endogenous ligand for TLR4 that is secreted from CaP cells and promotes inflammation. Inhibition of Prx1 by CaP cells resulted in reduced expression of VEGF, diminished tumor vasculature and retarded tumor growth. The mechanism by which Prx1 regulates VEGF expression in normoxic conditions was investigated in the current study. Our results show that incubation of mouse vascular endothelial cells with recombinant Prx1 caused increases in VEGF expression that was dependent upon TLR4 and required hypoxia inducible factor-1 (HIF-1) interaction with the VEGF promoter. The induction of VEGF was also dependent upon NF-κB; however, NF-κB interaction with the VEGF promoter was not required for Prx1 induction of VEGF suggesting that NF-κB was acting indirectly to induce VEGF expression. The results presented here show that Prx1 stimulation increased NF-κB interaction with the HIF-1α promoter, leading to enhanced promoter activity and increases in HIF-1α mRNA levels, as well as augmented HIF-1 activity that resulted in VEGF expression. Prx1 induced HIF-1 also promoted NF-κB activity, suggesting the presence of a positive feedback loop that has the potential to perpetuate Prx1 induction of angiogenesis. Strikingly, inhibition of Prx1 expression in CaP was accompanied with reduced expression of HIF-1α. The combined findings of the current study and our previous study suggest that Prx1 interaction with TLR4 promotes CaP growth potentially through chronic activation of tumor angiogenesis.

In recent years a number of studies have implicated chronic inflammation in prostate carcinogenesis. However, mitigating factors of inflammation in the prostate are virtually unknown. Toll-like receptor 4 (TLR4) activity is associated with inflammation and is correlated with progression risk in prostate cancer (CaP). TLR4 ligands include bacterial cell wall proteins, danger signaling proteins, and intracellular proteins such as heat shock proteins and peroxiredoxin 1 (Prx1). Here we show that Prx1 is overexpressed in human CaP specimens and that it regulates prostate tumor growth through TLR4-dependent regulation of prostate tumor vasculature. Inhibiting Prx1 expression in prostate tumor cells reduced tumor vascular formation and function. Furthermore, Prx1 inhibition reduced levels of angiogenic proteins such as VEGF within the tumor microenvironment. Lastly, Prx1-stimulated endothelial cell proliferation, migration, and differentiation in a TLR4- and VEGF-dependent manner. Taken together, these results implicate Prx1 as a tumor-derived inducer of inflammation, providing a mechanistic link between inflammation and TLR4 in prostate carcinogenesis. Our findings implicate Prx1 as a novel therapeutic target for CaP., (©2011 AACR.)

Previous studies have shown that tumor progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model is characterized by global DNA hypomethylation initiated during early-stage disease and locus-specific DNA hypermethylation occurring predominantly in late-stage disease. Here, we utilized Dnmt1 hypomorphic alleles to examine the role of Dnmt1 in normal prostate development and in prostate cancer in TRAMP. Prostate tissue morphology and differentiation status was normal in Dnmt1 hypomorphic mice, despite global DNA hypomethylation. TRAMP; Dnmt1 hypomorphic mice also displayed global DNA hypomethylation, but were characterized by altered tumor phenotype. Specifically, TRAMP; Dnmt1 hypomorphic mice exhibited slightly increased tumor incidence and significantly increased pathological progression at early ages and, conversely, displayed slightly decreased tumor incidence and significantly decreased pathological progression at advanced ages. Remarkably, hypomorphic Dnmt1 expression abrogated local and distant site macrometastases. Thus, Dnmt1 has tumor suppressor activity in early-stage prostate cancer, and oncogenic activity in late stage prostate cancer and metastasis. Consistent with the biological phenotype, epigenomic studies revealed that TRAMP; Dnmt1 hypomorphic mice show dramatically reduced CpG island and promoter DNA hypermethylation in late-stage primary tumors compared to control mice. Taken together, the data reveal a crucial role for Dnmt1 in prostate cancer and suggest that Dnmt1-targeted interventions may have utility specifically for advanced and/or metastatic prostate cancer.

To present representative data of long-term survivorship and clinical outcome for the PFC total knee arthroplasty (PFC-TKA). A consecutive series of 141 TKA was followed for a mean of 13 years (range, 11-16 years). Sixty-five knees were evaluated, 30 of these clinically and radiographically. Twenty-eight knees could only be assessed with the use of a questionnaire. Six patients were living in nursing homes. Fifty-four patients (65 knees) had died. Eleven had undergone a revision. One patient was considered lost to follow-up. With re-operation for any reason as the endpoint, the 10-year survival rate was 92% (n = 91 patients at risk), and the 14-year survival rate was 91% (n = 12). With aseptic loosening of the implant as the endpoint, the 10- and 14-year survival rates were 97%. The mean Knee Society and function scores were 76 and 48 points, respectively. In this multi-surgeon series modular fixed-bearing TKA had good clinical and radiographic results with excellent long-term survivorship.

The objective of this work is to find out how reliably knee joint alignment can be measured from a standardized photograph and what influence changes in the standing position have on the angles measured. The interrater, intrarater, and test-retest reliability were evaluated. The influence of image-object distance, the distance between the legs and leg rotation on the measured angles was evaluated. In addition to the digital photographs, 10 full-length radiographs were obtained in an upright position to determine whether the measured angles represent the anatomic axis or mechanical axis. There was high correlation between the interrater (ICC 0.997), intrarater (ICC 0.989) and test-retest reliability (ICC 0.904). Only slight deviation was found with the changes in radiograph-object distance (0 degrees -1.8 degrees ). With feet together varus malalignment was greater. Leg rotation showed a strong influence on the measured results (ICC 0.658). The angle measured in the digital photographs reflects the mechanical axis with only slight deviation (0.12 degrees -1.9 degrees ). The measurement of the clinical axis using standardized radiography is highly reliable and can be used for individual follow-up of varus and valgus malalignments.

We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system.

Aberrant DNA methylation plays a significant role in nearly all human cancers and may contribute to disease progression to advanced phenotypes. Study of advanced prostate cancer phenotypes in the human disease is hampered by limited availability of tissues. We therefore took advantage of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model to study whether three different phenotypes of TRAMP tumors (PRIM, late-stage primary tumors; AIP, androgen-independent primary tumors; and MET, metastases) displayed specific patterns of CpG island hypermethylation using Restriction Landmark Genomic Scanning. Each tumor phenotype displayed numerous hypermethylation events, with the most homogeneous methylation pattern in AIP and the most heterogeneous pattern in MET. Several loci displayed a phenotype-specific methylation pattern; the most striking pattern being loci methylated at high frequency in PRIM and AIP but rarely in MET. Examination of the mRNA expression of three genes, BC058385, Goosecoid, and Neurexin 2, which exhibited nonpromoter methylation, revealed increased expression associated with downstream methylation. Only methylated samples showed mRNA expression, in which tumor phenotype was a key factor determining the level of expression. The CpG island in the human orthologue of BC058385 was methylated in human AIP but not in primary androgen-stimulated prostate cancer or benign prostate. The clinical data show a proof-of-principle that the TRAMP model can be used to identify targets of aberrant CpG island methylation relevant to human disease. In conclusion, phenotype-specific hypermethylation events were associated with the overexpression of different genes and may provide new markers of prostate tumorigenesis.

The antitumor effects of 1,25-dihydroxyvitamin D(3) (calcitriol) are being exploited for prevention and treatment of prostate cancer (CaP). These studies examined the antiproliferative effects of calcitriol in primary cell cultures derived from transgenic adenocarcinoma of mouse prostate (TRAMP) mice chronically treated with calcitriol (20 microg/kg) or vehicle 3x/week from 4 weeks-of-age until palpable tumors developed. This is a report on the response of two representative control (Vitamin D naïve, naïve) and calcitriol-treated (Vitamin D insensitive, VDI) cells to calcitriol. VDI cells were less sensitive to calcitriol based on less cell growth inhibition and less inhibition of DNA synthesis as measured by MTT and BrdU incorporation assays. Similarly, VDI cells were less sensitive to growth inhibition by the vitamin analog, 19-nor-1alpha,25-dihydroxyvitamin D(2) (paricalcitol). There was no change in apoptosis following treatment of naïve and VDI cells with calcitriol. Vitamin D receptor (VDR) expression was up-regulated by calcitriol in both naïve and VDI cells. In addition, calcitriol induced the Vitamin D metabolizing enzyme, 24-hydroxylase (cyp24) mRNA and enzyme activity similarly in naïve and VDI cells as measured by RT-PCR and HPLC, respectively. In summary, VDI cells are less responsive to the antiproliferative effects of calcitriol. Understanding Vitamin D insensitivity will further clinical development of Vitamin D compounds for prevention and treatment of CaP.

Objectives: To assess the early histological effects of pharmacological androgen deprivation (AD), which have been assessed only over longer periods, as surgical castration leads rapidly to diminished cell proliferation and enhanced cell death within the prostate., Patients and Methods: With Institutional Review Board approval, 35 patients were randomly assigned (seven in each group) to receive 0, 7, 14, 21 and 28 days of AD (flutamide, 250 mg orally three times/day, and one injection with leuprolide acetate 7.5 mg) before radical prostatectomy. The surgical specimens were assessed by conventional histology and immunohistochemistry, while macroarray analysis and quantitative real-time polymerase chain reaction (QRT-PCR) were used to examine gene expression., Results: There were morphological changes within the prostatic tissues as early as 7 days after initiating AD, similar to the response to castration. There was tumour cell vacuolization indicating cellular injury, glandular atrophy and mononuclear cell infiltration as prominent and progressive effects but, by contrast with castration studies, there were no changes in epithelial proliferation or apoptosis. Macroarray analysis, validated by QRT-PCR and immunohistochemistry, showed up-regulation of numerous and potentially counter-effective genes involved in the cell cycle and apoptosis., Conclusions: Pharmacological AD induces significant involution within prostatic tissues over 7-28 days, but allows the persistence of some viable tumour cells capable of proliferation.

We examined the DNA methylation pathway in an autochthonous murine prostate cancer model, transgenic adenocarcinoma of mouse prostate (TRAMP). We observed that, compared with strain-matched normal prostates, primary and metastatic TRAMP tumors display increased cytosine DNA methyltransferase (Dnmt) activity, Dnmt1 and Dnmt3b protein expression, and Dnmt1, Dnmt3a, and Dnmt3b mRNA expression. Increased expression of Dnmt genes correlates with increased expression of cyclin A and E2F target genes, implicating increased cell proliferation and Rb inactivation in Dnmt overexpression. We analyzed DNA methylation in TRAMP and found that global levels of 5-methyl-2'-deoxycytidine are unaltered, whereas specific tumors display centromeric repeat hypomethylation. To interrogate locus-specific methylation, we did restriction landmark genomic scanning (RLGS) on normal prostates and primary tumors. In primary tumors, 2.3% of approximately 1,200 analyzed loci display aberrant DNA hypermethylation, whereas a considerably smaller number of events show hypomethylation. The pattern of RLGS changes was nonrandom, indicating a coordinated methylation defect. Two specific genes identified by RLGS were studied in detail. Surprisingly, methylation of a downstream exon of p16(INK4a) (p16) was the highest frequency hypermethylation event identified in TRAMP, where it is associated with increased p16 mRNA and protein expression. In contrast, hypermethylation of the 5' CpG island region of the homeobox gene Irx3 in TRAMP is associated with reduced gene expression. In summary, our data reveal a systemic DNA methylation pathway defect in TRAMP reminiscent of human prostate cancer, supporting the use of this model to investigate the functional role of DNA methylation pathway alterations in prostate cancer development.

Androgen has been implicated as a negative regulator of host immune function and a factor contributing to the gender dimorphism of autoimmunity. Conversely, androgen deprivation has been suggested to potentiate male host immunity. Studies have shown that removal of androgen in postpubertal male mice produces an increase in size and cellularity of primary and peripheral lymphoid organs, and enhances a variety of immune responses. Yet, few details are known about the effect of androgen removal on T cell-mediated immunity. In this study, we demonstrate two pronounced and independent alterations in T cell immunity that occur in response to androgen deprivation, provided by castration, in postpubertal male mice. First, we show that levels of T cells in peripheral lymphoid tissues of mice are increased by androgen deprivation. Second, T cells from these mice transiently proliferate more vigorously to TCR- and CD28-mediated costimulation as well as to Ag-specific activation. In addition, androgen deprivation accelerates normalization of host T and B cell levels following chemotherapy-induced lymphocyte depletion. Such alterations induced by androgen deprivation may have implications for enhancing immune responses to immunotherapy and for accelerating the recovery of the immune system following chemotherapy.

Background: Fatigue is one of the most frequent symptoms in cancer patients. It is the aim of medical and psychosocial rehabilitation programs to reduce physical impairments and psychosocial distress. There are no reliable data on the prevalence or the pattern of fatigue in patients attending such rehabilitation programs. We therefore initiated a cross-sectional study to evaluate this phenomenon., Patients and Methods: Using the EORTC QLQ-C30, HADS and MFI questionnaires in 144 patients with different malignant diseases the incidence, patterns and necessity of treatment of fatigue were evaluated., Results: One third of the patients were diagnosed with breast cancer, 27% with gastrointestinal cancers and 20% suffered from haematologic/lymphatic diseases. There was a good correlation between fatigue levels in the 3 instruments used. 22% of all patients showed significant symptoms of fatigue. The univariate analysis did not reflect any significant differences in severity of fatigue with respect to diagnosis, haemoglobin levels, medical and therapeutic history or sociodemographic data. However, using cluster analysis we could identify 3 groups of patients that differ significantly in fatigue and quality-of-life measurements., Conclusion: We conclude from our data that fatigue is an important phenomenon in cancer patients who attend medical and psychosocial rehabilitation programs. This has an impact on the ability and degree to reintegrate these patients into everyday life., (Copyright 2003 S. Karger GmbH, Freiburg)

Manipulations capable of breaking host tolerance to induce tissue-specific T cell-mediated inflammation are of central importance to tumor immunotherapy and our understanding of autoimmunity. We demonstrate that androgen ablative therapy induces profuse T cell infiltration of benign glands and tumors in human prostates. T cell infiltration is readily apparent after 7-28 days of therapy and is comprised predominantly of a response by CD4+ T cells and comparatively fewer CD8+ T cells. Also, T cells within the treated prostate exhibit restricted TCR Vbeta gene usage, consistent with a local oligoclonal response. Recruitment/activation of antigen-presenting cells in treated prostate tissues may contribute to local T cell activation. The induction of T cell infiltration in prostate tissues treated with androgen ablation may have implications for the immunotherapeutic treatment of prostate cancer as well as other hormone-sensitive malignancies, including breast carcinoma.

The production of B lymphocytes is regulated in part by physiologic levels of androgens and estrogens. While these sex hormones down-regulate B lymphopoiesis, augmentation of B lymphopoiesis occurs under conditions where androgen or estrogen levels are decreased. In this study we examine the effect of androgen ablation of male mice on B lymphopoiesis and on the phenotypic composition of peripheral B lymphocyte populations. Spleen and thymic weights are significantly increased following castration, as is the total number of peripheral blood lymphocytes. However, the absolute numbers of B cells in the periphery are selectively increased following castration; the numbers of T cells, NK cells and granulocytes remain unchanged. The increase in circulating B cells is due largely to increases in the numbers of recent bone marrow emigrants expressing a B220(lo+)CD24(hi+) phenotype and these cells remain significantly elevated in castrated mice for up to 54 days post-castration. Similar increases in the percentages of newly emigrated B cells are observed in mice that lack a functional androgen receptor (TFM:). Finally, assessments of B cell progenitors in the bone marrow revealed significant increases in the relative numbers of IL-7-responsive B cell progenitors, including cells in Hardy fractions B (early pro-B cells), C (late pro-B cells), D (pre-B cells) and E (immature B cells). These findings demonstrate that androgen ablation following castration significantly and selectively alters the composition of peripheral B cells in mice. Further, these alterations result from the potentiating effects of androgen ablation on IL-7-responsive pro-B cell progenitors.

Purpose: To evaluate the effect of the synthetic matrix metalloproteinase inhibitor, Galardin, on proteases produced by Pseudomonas aeruginosa (PA) and on a rabbit model of Pseudomonas keratitis., Methods: Protease activities of culture broths from Pseudomonas strains PA-28 and W-186 were characterized in vitro by gelatin zymography and by digestion of Azocasein in the presence and absence of Galardin and the serine protease inhibitor, aprotinin. In a noninfectious in vivo experiment, sterile PA culture broth from W-186 was injected intrastromally into rabbit corneas that were treated topically with Galardin or vehicle, then evaluated clinically and histologically. In an infectious in vivo experiment, rabbit corneas were injected with washed PA-28, then treated topically with Galardin or vehicle and clinically scored., Results: Gelatin zymography of culture broth from W-186 and PA-28 detected two proteases that were both inhibited by Galardin. Galardin reduced the digestion of Azocasein by both PA culture broths by 99%, whereas aprotinin did not significantly reduce the protease activity of PA-28 conditioned broth. Intrastromal injection of sterile W-186 culture broth caused rapid corneal destruction that was prevented by topical treatment with Galardin. Intrastromal injection of washed PA-28 bacteria resulted in progressive corneal melting that was significantly (P < 0.005) delayed, but ultimately not prevented, by topical treatment with Galardin., Conclusions: Pseudomonal protease activity in culture broth consisted predominantly of metalloproteinases and were effectively inhibited by Galardin in vitro. Topical treatment with Galardin prevented destruction of rabbit corneas by bacterial products present in culture broth, and it delayed corneal destruction after injection of PA bacteria. Galardin may be a useful adjuvant when corneal destruction proceeds despite prompt antibiotic treatment.

In Experiment 1, 12 unmated cyclic ewes received twice-daily intrauterine injections on Days 12 to 14 of one of the following treatments: 1) ovine conceptus secretory proteins (oCSP) containing 25 microg of ovine trophoblast protein-1 (oTP-1) as determined by RIA; 2) 25 or 50 microg recombinant human interferon alpha1 (rhlFN); or 3) 1500 microg of serum proteins (oSP) from a Day-16 pregnant ewe (estrus = Day 0) per uterine horn. Ewes receiving oCSP had longer interestrous intervals (27 +/- 2 days; P<0.05) than ewes receiving oSP (17 +/- 2 days). Ewes receiving either dose of rhlFN had an interestrous interval of 16 +/- 2 days which did not differ (P>0.10) from that of oSP-treated ewes. In Experiment 2, 59 normally cycling ewes, mated on Day 0, received twice-daily intramuscular injections of either 2 mg recombinant bovine interferon alpha1 (rblFN) or placebo on Days 12 to 15 post estrus. On Day 16, pregnancy was confirmed by flushing a morphologically normal conceptus from the uterus. Pregnancy rates for rblFN-treated (80%) and placebo-treated (62%) ewes were not different (P>0.10). Uterine flushings and conceptus-conditioned medium were assayed for oTP-1. Total oTP-1 in conceptus-conditioned culture medium was higher (P<0.02) when conceptuses were from placebo-treated (104 +/- 14 microg/conceptus) than from rblFN-treated (56 +/- 12 microg/conceptus) ewes; while total oTP-1 in uterine flushings was similar (P>0.10) for placebo-treated (132 +/- 15 microg/conceptus) and rblFN-treated (147 +/- 17 microg/conceptus) ewes. The interval from mating to subsequent estrus following conceptus removal was 31 +/- 1 and 28 +/- 1 days for pregnant ewes treated with rblFN and placebo, respectively. Interestrous intervals for nonpregnant ewes were longer (P<0.02) for rblFN-treated (27 +/- 3 days) than for placebo-treated (18 +/- 2 days) ewes.

This study was conducted to determine whether intrauterine infusion of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1), which has 70% sequence identity to bovine trophoblast protein-1, will prevent regression of corpora lutea anticipated to have a short lifespan. Twenty-six beef cows in good body condition were allotted to four treatment groups at parturition in a 2 x 2 factorial design. Treatments were: group 1, saline; group 2, rboIFN-alpha I1; group 3, norgestomet-saline; and group 4, norgestomet-rboIFN-alpha I1. Norgestomet implants were inserted on days 21-24 postpartum and removed 9 days later (before injection of human chorionic gonadotrophin (hCG)). Ovulation was induced 30 to 33 days postpartum with 5000 or 10,000 iu hCG. Groups 1 (n = 7) and 3 (n = 5) were given intrauterine infusions (rectocervical approach) twice daily with saline on days 1-12 or 13-24 after hCG injection, respectively. Cows allotted to groups 2 (n = 8) and 4 (n = 6) were given intrauterine infusions (rectocervical approach) of 2 mg rboIFN-alpha I1 twice daily on days 1-12 or 13-24 after hCG injection, respectively. Treatment with both norgestomet and rboIFN-alpha I1 delayed (P less than 0.01) luteolysis. Lengths of luteal phases (days; mean +/- SEM) were 8.4 +/- 0.7 (group 1, saline), 14.1 +/- 1.0 (group 2, rboIFN-alpha I1), 18.6 +/- 1.3 (group 3, norgestomet-saline) and 20.8 +/- 1.2 (group 4, norgestomet-rboIFN-alpha I1). Concentration of progesterone in serum was similar among all groups the first 6 days following hCG-induced ovulation, but differed (P less than 0.01) thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was conducted to determine if charcoal-extracted follicular fluid inhibits FSH-induced follicular development in prepuberal heifers. Thirty-six prepuberal heifers were allotted by breed and weight to a 2 x 2 factorial experiment involving charcoal-extracted follicular fluid and FSH treatments. Heifers were unilaterally ovariectomized and injected (intravenously; 10 ml) every 8 h for 88 h with either charcoal-extracted follicular fluid or saline. Follicle-stimulating hormone (2 mg) or saline was injected (intramuscularly) every 8-h starting 24 h after initiation of charcoal-extracted follicular fluid to 88 h following unilateral ovariectomy. Plasma samples were collected at 8-h intervals from 48 h prior to unilateral ovariectomy to 96 h following unilateral ovariectomy when the remaining ovary was removed. Follicular fluid and total ovarian weight increased following FSH treatment. The increases were not inhibited by charcoal-extracted follicular fluid. Total number of surface follicles was similar among treatments. However, FSH induced a shift in follicular diameter from small (less than or equal to 3 mm) to medium (7 to 9 mm) or large (10 to 13 mm) follicles, which was unaffected by charcoal-extracted follicular fluid. Plasma concentration of FSH, but not LH, declined following charcoal-extracted follicular fluid administration. In summary, charcoal-extracted follicular fluid did not inhibit FSH-induced follicular development in prepuberal heifers when charcoal-extracted follicular fluid was administered at a dosage that reduced circulating concentration of FSH by approximately 40%.