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Background: Two features of the progression from organ-confined to metastatic prostate cancer are dysregulation of the androgen receptor (AR) and a decrease in insulin-like growth factor-type-I receptor (IGF-IR) expression. The purpose of this study was to determine the effect of changes in IGF-IR expression on AR activity., Methods: M12 human prostate cells were stably transfected with an AR expression construct to produce the M12-AR parental (PAR) cell line. PAR cells were implanted orthotopically into nude mice and M12-AR primary (PRI) cell lines were derived from intraprostatic tumors and metastatic cell lines (MET) were derived from PRI tumors that had metastasized to diaphragm or lung., Results: Tumor formation in the prostate by PAR cells was decreased significantly compared to M12 controls. PAR, PRI, and MET cells expressed equivalent amounts of AR protein; however, IGF-IR expression was increased significantly in PAR and PRI cells. IGF-IR expression decreased in MET lines to the levels seen in M12 control cells. IGF-I significantly enhanced dihydrotestosterone (DHT)-stimulated, but not basal, AR transcriptional activity in PRI cells. In MET cells, IGF-I significantly suppressed DHT-stimulated transcriptional activity. In MET cells in which the IGF-IR was re-expressed from a retroviral vector, the effects of DHT and IGF-I on AR activity were similar to those seen in PRI cells., Conclusions: This study demonstrates that the changes in IGF-IR expression exhibited by this model of metastatic progression cause significant alterations in AR signaling and suggest that this interaction may be an important aspect of the changes seen in AR function in disease progression in vivo., (2004 Wiley-Liss, Inc.)

Overexpression of mac25 in the prostate cancer cell line M12 effects a dramatic reversal of the transformed phenotype. cDNA array analysis of RNA from cells overproducing the mac25 protein (M12/mac25) indicated upregulation of the sex determining transcription factor SOX9. In this study, we have confirmed increased expression of SOX9 in M12/mac25 cells and have further investigated the physiological effects of increased SOX9 production. Greatly increased levels of SOX9 RNA and mature protein were demonstrated in cells transfected with a SOX9 cDNA (M12/SOX9), and gel mobility shift assays confirmed binding of nuclear protein from these cells to an oligonucleotide containing the SOX9 consensus binding sequence. M12/SOX9 cells assumed the spindle-shaped morphology characteristic of M12/mac25 cells, suggesting that SOX9 mediates some effects of mac25. Elevated expression of SOX9 resulted in a decreased rate of cellular proliferation, cell cycle arrest in G0/G1, and increased sensitivity to apoptosis. Tumor development in athymic nude mice was inhibited by 80%. Finally, prostate-specific antigen and the androgen receptor, two genes whose expression is characteristic of differentiated cells, were both upregulated in M12/SOX9 cells. These data indicate that SOX9 contributes to growth regulation by mac25 via inhibition of cell growth and promotion of differentiation.

Background: In the prostate, insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1/mac 25) appears to be decreased in cancer. Likewise, mice injected with prostate cells over-expressing IGFBP-rP1/mac 25 showed decreased tumor formation and tumor volume., Methods: Immunohistochemistry was used to determine the presence of IGFBP-rP1/mac 25 in human prostate tissue and in LuCaP 23.12 prostate cancer xenografts., Results: Immunoreactivity for IGFBP-rP1/mac 25 was detected in the cytoplasm and nuclei of benign prostate epithelium. Cancer cells also stained but the level of intensity was less than that observed in benign epithelium (P < 0.05). Within the stroma, peripheral nerves, and endothelial vessels reacted with IGFBP-rP1/mac 25 antibodies. IGFBP-rP1/mac 25 was also observed in the nuclei of LuCaP 23.12 cells. LuCaP xenografts with smaller tumor volumes demonstrated more staining, and larger tumor volumes had less staining (P = 0.0033, R = 0.610). There was also a significant inverse correlation between IGFBP-rP1/mac 25 levels in LuCaP 23.12 cells and the proliferative index, measured by percent of cells staining for 5-bromo-2'-deoxyuridine (Br-dU) (P = 0.0045, R = 0.594)., Conclusions: IGFBP-rP1/mac 25 is present in normal prostate epithelium and is decreased in human prostate malignancy and higher IGFBP-rP1/mac 25 levels are associated with reduced tumor growth., (Copyright 2003 Wiley-Liss, Inc.)

Increased expression of mac25/insulin-like growth factor binding-protein related protein-1 (IGFBP-rP1) in human breast and prostate epithelial cell lines results in the suppression of tumor growth. CDNA expression array analysis revealed increased manganese superoxide dismutase (SOD-2) expression in the mac25/IGFBP-rP1-transfected M12 human prostate cancer cell line compared to M12 control cells. SOD-2 has been postulated to be a tumor suppressor. SOD-2 was also increased in LNCaP cells stably transfected with mac25/IGFBP-rP1, but not in mac25/IGFBP-rP1-transfected PC-3 cells. Mac25 LNCaP cells had a marked decrease in tumor growth in nude mice compared to controls, but there was no difference in tumor growth in mac25 PC-3 cells compared to control. Phosphorylated Erk and Akt were increased in the M12 and LNCaP transfected mac25/IGFBP-rP1 cells but not PC-3 mac25. Inhibition of PI-3 kinase results in a marked decrease in viability of the M12-mac25 cells compared to M12 controls. Cells treated with H(2)O(2) result in an increase in phospho-ERK. Transfection of SOD-2 in M12 cells markedly decreased tumor growth, apoptosis, G1 delay in the cell cycle, and expression of senescence associated beta-galactosidase. These results suggest that one of the downstream mediators of the senescence-associated tumor suppression effect of mac25/IGFBP-rP1 is SOD-2.

Objective: Human papillomavirus (HPV) assays are likely to be used with increasing frequency in clinical management of women with abnormal Papanicolaou smears and in cervical cancer screening. Our objective was to simplify the method of collection of female genital tract specimens. The utility of vaginal dry swabs for HPV diagnosis was evaluated., Methods: Specimens for cytology and for HPV identification were collected by a clinician from 189 female soldiers attending a military clinic. Three methods of specimen collection for HPV identification were compared: a vaginal dry swab (v-DRY), and vaginal and cervical swabs placed into specimen transport medium (v-STM and c-STM). Swabs were shipped to a STD laboratory for processing. Specific HPV types were identified by a consensus primer based PCR based method. Results from 165 women were evaluable., Results: HPV prevalence by the three methods was similar and ranged from 44.8% to 50.9%. 53 (32.1%) women were HPV positive and 60 (36.4%) women were HPV negative by all three collection methods. With respect to the risk categories of specific HPV types, there was greater agreement between the results from the two vaginal (v-DRY and v-STM) samples (kappa values of 0.69-0.81) than between the cervical (c-STM) and either of the vaginal samples (kappa values of 0.37-0.55). The HPV yield from c-STM was somewhat greater than that from the vaginal specimens but the correlation between cytological abnormalities and HPV was high for all three methods., Conclusion: A dry vaginal swab may be an acceptable method of specimen collection for HPV diagnosis.

Insulin-like growth factors (IGFs) and the type 1 IGF receptor (IGF-R) are involved in normal growth and development of the human prostate. Changes in levels of IGF-R and IGFs have been shown for several malignancies. Immunohistochemistry and in situ hybridization were performed to compare the expression of IGF-R and IGF-II in vivo in prostate tissue containing benign epithelium, high grade prostate intraepithelial neoplasia (PIN), and adenocarcinoma. Messenger ribonucleic acid (mRNA) hybridization signals and immunoreactivity for IGF-R were localized primarily to epithelial cells, with less signal in stroma. IGF-R mRNA was significantly decreased by 42% in PIN and 35% in cancer cells compared to that in benign epithelium (P < 0.0001). IGF-R immunostaining was significantly decreased by 32% in PIN and by 42% in malignant epithelium compared to that in benign epithelium (P < 0.004). IGF-II mRNA was also localized primarily to epithelial cells. IGF-II mRNA was significantly increased by 30% in adenocarcinoma compared to that in benign epithelium (P < 0.03). Immunoreactivity for IGF-II was localized to both stroma and epithelium. Protein levels for IGF-II were not significantly increased in cancer cells compared to those in benign epithelium. The decrease in the type 1 IGF receptor and increase in IGF-II mRNA may affect prostate cancer proliferation and differentiation.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) modulate the actions of IGF. We have previously reported that IGFBP-2 messenger ribonucleic acid (mRNA) and protein are increased, and IGFBP-3 protein decreased in malignant prostate epithelium compared to benign epithelium. In this study, we examined the other IGFBPs secreted by prostate cells in vitro, namely IGFBP-4, -5, and 6. Immunoreactivity and mRNA signals for IGFBP-4 and -6 were localized to epithelial cells, with less signal in stroma. IGFBP-4 immunostaining and hybridization signal were significantly increased in prostate adenocarcinoma compared to those in benign epithelium. Immunostaining for IGFBP-5 was localized to the epithelium and stroma. IGFBP-5 immunoreactivity was significantly increased in malignant compared to benign epithelium. IGFBP-5 mRNA signal was not localized to epithelial cells; rather, the signal was over stromal cells surrounding the acinar structures. These cells are thought to be fibroblasts. We show that IGFBP-4 mRNA and protein and IGFBP-5 protein are increased in malignant epithelium compared to benign epithelium, that IGFBP-6 is present in benign and malignant epithelium, and that there is differential localization of IGFBP-5 mRNA and protein in prostate tissue. IGFBP-5 that is made by fibroblasts appears to be sequestered by epithelial cells. IGFBP-5 may, therefore, be a factor in cellular interactions between stromal and epithelial cells that are of fundamental importance for normal prostatic development and function.

Purpose: The insulin-like growth factor system has recently been shown to have important mitogenic effects in the prostate. The system consists of 2 ligands: insulin-like growth factor types 1 and 2, which are potent mitogens for prostate cell growth, and 6 insulin-like growth factor binding proteins that modify the activity of these growth factors. Recently, changes in serum levels of insulin-like growth factor binding proteins 2 and 3 have been identified in prostate cancer patients but in vivo studies of these changes have not been previously reported., Materials and Methods: We performed immunohistochemical staining for insulin-like growth factor binding proteins 2 and 3 in areas of benign disease, prostatic intraepithelial neoplasia, and cancer from specimens obtained from 24 patients who underwent prostatectomy to determine changes in the quantity of these proteins with progression from the benign through the premalignant and into the malignant states. Quantification of the differences noted in the amount of insulin-like growth factor binding proteins 2 and 3 found in each of the 3 tissue types was then correlated with serum and tissue prostate specific antigen levels, pathological stage and grade of the tumors., Results: Our data indicate that as human prostate tissue progresses from the benign to the malignant state, insulin-like growth factor binding protein 2 immunoreactivity in the prostatic luminal epithelial cells increases and that of insulin-like growth factor binding protein 3 decreases. Quantification of the differences noted in binding proteins 2 and 3 among all 3 tissue types was statistically significant. However, no correlation was noted between the stage or grade of prostate carcinoma and insulin-like growth factor binding protein 2 or 3 immuno-staining intensity. Additionally, no significant correlation was found between serum or tissue levels of prostate specific antigen and insulin-like growth factor binding protein 2 or 3 immunoreactivity., Conclusions: Our data suggest that significant changes in the insulin-like growth factor system occur with malignant transformation in the prostate. It is hoped that this system may eventually result in a better serum or tissue (from prostate biopsy) marker of malignant transformation, and/or represent a target for early therapeutic intervention to alter the growth and development of prostate cancer.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) modulate the activity of IGFs. In vitro human prostate epithelial cells secrete IGFBP-2 and -3. In vivo IGFBP-2 is increased, and IGFBP-3 is decreased in the serum of patients with prostate cancer. Immunohistochemistry and in situ hybridization were performed to compare the expression of IGFBP-2 and -3 in vivo in prostate tissue containing benign epithelium, high grade prostate intraepithelial neoplasia (PIN), and adenocarcinoma. Immunoreactivity and messenger ribonucleic acid (mRNA) hybridization signals for IGFBP-2 and -3 were localized to epithelial cells. IGFBP-2 immunostaining intensity was significantly increased in PIN regions compared to that in normal epithelium and was further increased in malignant cells. IGFBP-2 mRNA was also significantly increased in PIN and cancer cells. IGFBP-3 immunoreactivity was significantly increased in PIN regions compared to normal epithelium; however, IGFBP-3 protein was significantly decreased in malignant cells. IGFBP-3 mRNA remained virtually unchanged in benign epithelium, PIN, and adenocarcinoma cells. These results demonstrate that increased IGFBP-2 protein in PIN and malignant cells is probably due to increased mRNA expression. However, levels of IGFBP-3 protein may be due to pre- and/or posttranslational mechanisms, including proteolysis. The changes in IGFBP-2 and -3 protein levels in prostatic tissue are in agreement with serum changes reported in patients with prostate cancer.

Chlorotriazines are widely used in agriculture as broadleaf herbicides. The compounds specifically inhibit photosynthesis, and, as such, display little interaction with animal systems. However, a 24-month feeding study with atrazine (ATR) revealed a significant dose-related increase of mammary tumors in female Sprague-Dawley (SD) rats. Because numerous studies indicated that ATR had a low mutagenic and oncogenic potential, it was decided to test a hypothesis that the herbicide possessed endocrine activity. Among tests for estrogenic action, oral dosing of ATR up to 300 mg/kg did not stimulate uterine weight of ovariectomized rats. However, ATR administration did reduce estrogen-stimulated uterine weight gain. Further evidence of inhibition came from measures of [3H]-thymidine incorporation into uterine DNA of ATR-treated immature rats. Again, no intrinsic estrogenic activity was observed up to a 300-mg/kg dose. In vitro, ATR competed poorly against estradiol binding to cytosolic receptors, with an approximate IC50 of 10(-5) M. Atrazine administration to SD and Fischer-344 (F-344) rats for 12 months, up to 400 ppm in food, was correlated with significant alterations of estrous cycling activity; but there was a divergent strain response. SD rats showed an increased number of days in vaginal estrus, increased plasma estradiol, and decreased plasma progesterone by 9 to 12 months of treatment. F-344 rats did not demonstrate treatment-related affects. A study of ultrastructure in the hypothalamic arcuate nucleus of female SD rats that were fed diaminochlorotriazine (DACT), an ATR metabolite, suggested that age-associated glial pathology was enhanced by treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Several published reports have indicated that certain chloro-s-triazine herbicides may alter endocrine function in rats, possibly by androgen receptor binding. In direct tests of estrogenic bioactivity, oral doses of up to 300 mg/kg/d of atrazine, simazine, or the common metabolite diaminochlorotriazine (DACT) did not significantly increase uterine weight of ovariectomized Sprague-Dawley female rats. The highest dose, which was approximately 10% of the LD50 for these compounds, did cause body weight loss. When administered concomitantly with sc injections of estradiol (2 micrograms/kg), 300 mg/kg of orally administered chlorotriazines significantly reduced uterine weight in comparison to animals given estrogen alone. Neither atrazine, simazine, nor DACT, at oral doses up to 300 mg/kg/d, stimulated incorporation of [3H]thymidine into uterine DNA of immature Sprague-Dawley female rats. However, oral treatment at doses of 50 mg/kg and higher significantly reduced thymidine incorporation into uterine DNA extracted from immature rats given a single injection of 0.15 microgram estradiol. Oral doses of 300 mg/kg of atrazine, simazine, or DACT significantly reduced expression of progesterone receptor binding in cytosol fractions prepared from uteri of ovariectomized rats injected sc with 1 microgram estradiol; 50 mg/kg triazine was not effective in this case. Uterine progesterone receptor levels were not stimulated in rats given oral doses up to 300 mg/kg of these triazines without estradiol injections. These results suggest that atrazine, simazine, and DACT possess no intrinsic estrogenic activity but that they are capable of weak inhibition of estrogen-stimulated responses in the rat uterus. This inhibition may play a role in the previously observed disruptive actions of chlorotriazines on reproductive endocrine function of female rats.

In an accompanying article (see pp. 183-196), it was reported that administration of very high doses of the chlorotriazine herbicides atrazine, simazine, and diaminochlorotriazine (DACT), a common metabolite, expressed antiestrogenic activity in uteri of female Sprague-Dawley rats without expressing intrinsic estrogenic activity. In the present article, studies of chlorotriazine interaction with rat uterine estrogen receptors (ER) are reported. Under equilibrium conditions, none of the triazine compounds showed an ability to compete against binding of radiolabeled estradiol to ER. A weak competition was evident only if cytosols were preincubated with triazines at 25 degrees C prior to introduction of tracer. Competition was very weak, with kl estimates of 10-100 microM. A limited Scatchard analysis suggested a competitive type of inhibition. Sucrose gradient analysis of cytosol incubations showed that triazine interaction with the 4S isoform of ER may be greater than with the 8S form. When administered to ovariectomized rats for 2 d at 300 mg/kg/d, atrazine, simazine, or DACT all reduced uterine ER binding capacity by approximately 30%. Results from the receptor binding studies indicated that triazine competition against ER binding occurred to a much lesser degree than inhibition of estrogen-mediated responses reported in accompanying articles. This suggests that the complete responses to triazines may include inhibition of events other than or in addition to ER binding of estrogen.