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Prochiral malonic diesters containing a quaternary carbon center have been successfully transformed into a diverse set of (t)Boc-Fmoc-α(2,2)-methyllysine-OH analogues through chiral malonic half-ester intermediates obtained via enzymatic (Pig Liver Esterase, PLE) hydrolysis. The variety of chiral half-ester intermediates, which vary from 1 to 6 methylene units in the side chain, are achieved in moderate to high optical purity and in good yields. The PLE hydrolysis of malonic diesters with various side chain lengths appears to obey the Jones's PLE model according to the stereochemical configurations of the resulting chiral half-esters. The established synthetic strategy allows the construction of both enantiomers of α(2,2)-methyllysine analogues, and a (S)-β(2,2)-methyllysine analogue from a common synthon by straightforward manipulation of protecting groups. Two different straightforward and cost effective synthetic strategies are described for the synthesis of α(2,2)-methyllysine analogues. The described strategies should find significant usefulness in preparing novel peptide libraries with unnatural lysine analogues. A Vapreotide analogue incorporating (S)-α(2,2)-methyllysine was prepared. However, the Vapreotide analogue with (S)-α-methyl-α-lysine is found to lose its specific binding to somatostatin receptor subtype 2 (SSTR2).

Purpose: Inhibition of neovessel development can stabilize tumor growth. A rapid in vitro method that can evaluate the effectiveness of anti-angiogenic drugs would aid in drug development. We tested a series of investigational agents to determine their ability to inhibit angiogenesis in our in vitro human angiogenesis model., Methods: A total of 74 neuroendocrine tumors were tested with five therapeutic agents for anti-angiogenic activity. Angiogenic responses were assessed visually and the percent of tumor explants that developed an angiogenic response was determined. The extent of neovessel growth was rated using a validated semi-quantitative visual scale. Analysis of variance was used to compare treatment outcome results to control values for these angiogenic parameters., Results: Vatalanib (2 × 10(-5) M) and patupilone (1 × 10(-8) M) were highly effective inhibitors of human tumor angiogenesis (mean overall angiogenic response for drug versus control 1.3 vs. 5.9 and 0.2 vs. 5.2, respectively) and were statistically significant at p <0.0001. Imatinib (2.5 × 10(-6) M) and everolimus (1 × 10(-8) M) were also effective (mean overall angiogenic response for drug versus control 2.2 vs. 5.9 and 4.5 vs. 5.9, respectively), and these were also statistically significant at p <0.0001. Pasireotide (1 × 10(-8) M) had no effect on angiogenesis (mean overall angiogenic response for drug vs. control 5.5 vs. 5.2)., Conclusions: Significant differences in angiogenic response to test drugs were noted in this neuroendocrine patient population. In vitro screening of a large series of fresh human tumors may be a cost-effective way to select drugs for continued clinical development.

Background: Only one tumor site is usually biopsied to determine the histologic features of that patient's entire tumor burden. We hypothesized that there are significant histologic and functional differences in primary neuroendocrine tumors (NETS) and their nodal or organ metastases. We also hypothesized that limited tumor sampling could lead to erroneous assumptions about the tumor's histologic characteristics and clinical behavior., Materials and Methods: Thirteen patients with metastatic well differentiated midgut NETS underwent simultaneous removal of their primary tumor, nodal metastasis, and organ metastasis. Each tumor site was stained quantitatively for Ki-67, chromogranin A (CGA), synaptophysin, CD31, and Factor VIII. Samples were also evaluated with in vitro tumor angiogenesis and drug chemoresistance assays., Results: Ki-67 staining was nearly identical at all sites tested. Quantitative stains for CGA, synaptophysin, cluster of differentiation 31 (CD31), and Factor VIII varied considerably among the patient's three tissue site samples. Only 6% of the tissue samples tested against a battery of chemotherapeutic agents exhibited susceptibility to a single drug at all three tumor sites. In contrast, several antiangiogenic agents exhibited uniform effectiveness across all three tissue sites in multiple patients., Conclusions: Sampling only one NET tumor site may lead to erroneous assumptions about the tumor's histologic features and functional behavior. Evaluation of primary tumors and their nodal and organ metastasis may be necessary to optimize clinical decision making., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: Proton pump inhibitors (PPIs) are used primarily to treat gastroesophageal reflux disease. Proton pump inhibitor-induced achlorhydria increases circulating gastrin and chromogranin A (CGA). Chromogranin is a widely used biomarker for the diagnosis and follow-up for gut-based neuroendocrine tumors (NETs). Proton pump inhibitor-induced increases in CGA or gastrin may falsely suggest the presence of a NET when none exists. Pancreastatin, a fragment of CGA, is also commonly used to diagnose and follow NETs. We hypothesized that chronic PPI use would increase circulating plasma gastrin, CGA, and pancreastatin levels., Methods: Thirty patients who used PPIs for 6 months or more (mean ± SD duration, 3.1 ± 2.5 years) and a separate control group of 30 patients who never used antacid medications were prospectively evaluated with plasma gastrin, CGA, and pancreastatin determinations., Results: Chronic PPI use resulted in significant increases in CGA (15.1 ± 11 vs 131 ± 207 ng/mL; P = 0.005) and significant increases in gastrin (34.8 ± 22.3 vs 167.8 ± 136.2 pg/mL; P = 0.001) compared to controls. In contrast, pancreastatin level in nonusers and chronic PPI users were identical (81.6 ± 36.4 vs 89.4 ± 43.4 pg/mL; P = 0.46)., Conclusions: Pancreastatin levels do not change with chronic PPI use and normal pancreastatin levels may be used to distinguish between drug-induced changes in biomarkers and tumor-related increases in circulating biomarkers.

Background: High doses (10 nM) of epothilone B, a microtubule stabilizer, will inhibit the development of human tumor-derived angiogenesis following short (14 d) drug exposure times. Metronomic dosing regimes use lower drug doses and prolonged drug exposure times in an attempt to decrease toxicity compared with standard dosing schedules. We hypothesized that epothilone B would be an effective anti-angiogenic agent when administered at very low doses over an extended period of time., Methods: Fragments of four fresh human tumors were cultured in a fibrin-thrombin matrix and maintained in nutrient media plus 20% fetal bovine serum (FBS) for 56 d. Tumor fragments (n=40-60 per group) were exposed to weekly doses of epothilone B at concentrations of 10, 5, 1, 0.5, or 0.1 nM. All of these concentrations are clinically achievable. Tumor angiogenesis was assessed weekly on d 14-56 using a validated visual grading system. This system rates neovessel growth, density, and length on a 0-16 scale [angiogenic index, (AI)]. The average change in AI between d 14 and 56 was calculated for all samples and used to evaluate the metronomic response., Results: Epothilone B produced a dose-dependent anti-angiogenic response in all tumors. Two of the four tumors demonstrated a clear and significant metronomic anti-angiogenic effect over time., Conclusions: Epothilone B, when dosed by a metronomic schedule may have a significant anti-angiogenic effect on human solid tumors. This study provides evidence for the potential use of epothilone B on a metronomic dosing schedule., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The first studies to assess in vitro angiogenesis in neuroendocrine tumors used animal-based assays to study the antiangiogenic properties of somatostatin analogs. Current technologies enable investigators to directly appraise the in vitro angiogenic response of an individual's neuroendocrine tumor with and without potential antiangiogenic reagents. This article describes the evolution of methods to assess in vitro angiogenesis in neuroendocrine tumors and describes some of the clinical data., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: In preclinical models, VEGF is a potent stimulant of both physiologic and pathologic angiogenesis. Conversely, anti-VEGF regimens have successfully inhibited angiogenesis both in vitro and in vivo. We hypothesized that VEGF would stimulate both physiologic and pathologic angiogenesis in a human-based fibrin-thrombin clot angiogenesis assay. We further speculated that anti-VEGF regimens would inhibit angiogenesis in this assay., Methods: To test these hypotheses, discs of human placental veins (physiologic model) and fragments of human tumors (pathologic model) were embedded in fibrin-thrombin clots and treated with either VEGF-A165 (VEGF) or anti-VEGF pathway reagents including bevacizumab, IMC-18F1, IMC-1121, and PTK787 (n=30 wells per treatment group, multiple concentrations tested in each specimen). Angiogenic responses were assessed visually using a previously validated grading scheme. The percent of tissue explants that developed angiogenic invasion into the clot (% I) as well as the extent of angiogenic growth (AI) via a semi-quantitative scale were assessed at set intervals., Results: VEGF failed to stimulate angiogenesis in both the physiologic and the pathologic model. While anti-VEGF reagents that targeted only one element of the VEGF pathway failed to consistently inhibit angiogenesis, PTK787, a receptor tyrosine kinase inhibitor that targets multiple VEGF and non-VEGF receptors, profoundly inhibited both physiologic and pathologic angiogenesis., Conclusion: These results suggest that VEGF-related pathways may not be solely responsible for stimulating angiogenesis in humans. Targeting the VEGF pathway in combination with elements of other growth factor pathways may provide a more effective means of inhibiting angiogenesis than targeting VEGF alone., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Objectives/hypothesis: AlloDerm (LifeCell Corp., Branchburg, NJ) is commonly employed for reconstruction of ablative soft tissue and mucosal defects following surgical resections. Although devoid of growth factors, AlloDerm may serve as an adhesive matrix for binding of growth factors, increasing local angiogenesis, and wound healing. We hypothesized that AlloDerm would enhance angiogenesis and might be altered with autologous blood products to enhance initiation of the angiogenic response., Methods: We used a human placental vein in a fibrin-thrombin clot-based angiogenesis model. Four groups, human placental vein (HPVM), HPVM with AlloDerm, HPVM with AlloDerm plus platelet-poor plasma, and HPVM with AlloDerm plus platelet-rich plasma were evaluated. Endothelial cell growth was evaluated visually (40x). Hematoxylin and eosin staining and immunofluorescent staining for growth within the AlloDerm matrix were also performed. To assess human umbilical vein endothelial cell (HUVEC) sites of attachment to AlloDerm, we incubated HUVEC cells with AlloDerm for a period of 2 weeks and evaluated attachment with anti-factor VIII immunofluorescence., Results: Angiogenic initiation decreased in the combined placental vein with AlloDerm group (P < .0001 at day 7, 14, 21). Additionally, initiation in the AlloDerm plus platelet-poor plasma group was significantly better than the AlloDerm alone group when placentas 2 and 3 were compared (P < .0001). On hematoxylin and eosin staining and immunofluorescent factor VIII staining, no endothelial growth into the AlloDerm was noted in the samples analyzed., Conclusions: AlloDerm may be enriched with platelet-poor plasma to stimulate greater initiation and wound healing; however, AlloDerm inhibits angiogenic initiation in this model.

Background: Well-differentiated, "typical" carcinoid tumors traditionally have a very poor response to chemotherapy. We hypothesized that tumor specimens from well-differentiated carcinoid tumors would be highly resistant to the effects of chemotherapy when tested against a variety of antineoplastic agents in vitro., Methods: Ninety-eight typical carcinoid specimens were surgically harvested, cultured, and tested against antineoplastics in vitro. (3)H-Thymidine incorporation was used to assess the percentage of cell-growth inhibition (PCI) of tested specimens. PCI was used to determine if specimens had extreme drug resistance (EDR), intermediate drug resistance (IDR), or low drug resistance (LDR) to each reagent against which they were tested., Results: Seventy specimens generated results. Each was tested with an average of six drugs. The mean proportions of drugs classified as LDR, IDR, and EDR were 0.48 (range 0-1), 0.34 (range 0-1), and 0.18 (range 0-0.80), respectively. The mean numbers of drugs per specimen exhibiting LDR, IDR, and EDR chemoresistance were 2.7, 2.1, and 1.2, respectively. 57 of 70 specimens (81%) had LDR to at least two drugs. 5-FU had the highest frequency of low chemoresistance at 69%, followed by doxorubicin at 67%. Low in vitro resistance to chemotherapeutics was prevalent among typical carcinoids, while EDR was comparatively infrequent., Conclusions: This implies that there may be less clinical chemoresistance and more chemosensitivity among typical carcinoid tumors than clinical trials have previously revealed. These findings warrant additional investigations assessing the response of carcinoid tumors to assay-guided chemotherapy regimens.

Background: Many cytotoxic drugs maintain antiangiogenic properties, but there are no human, tumor-based assays to evaluate their antiangiogenic potential. We used a fibrin-thrombin clot-based angiogenesis model to evaluate the angiogenic response of human breast cancer to various cytotoxic agents commonly used in its treatment., Methods: Fragments of freshly harvested human breast tumors were embedded in fibrin-thrombin clots and treated with five drugs: adriamycin, taxol, 5-fluorouracil (5-FU), methotrexate, and vincristine. Each treatment group included a mean of 28 fragments (range 16-60). A total of four tumors were tested. Tumor fragments were tested with a single dose of each reagent. Angiogenic initiation, angiogenic growth, and overall angiogenic effect were determined for each treatment group using a previously validated scale., Results: All four breast cancer specimens tested developed an angiogenic response, sprouting neovessels in vitro in a time-dependent fashion (r = 0.84, P = 0.0007). Taxol statistically inhibited angiogenesis in all four specimens with decreases in the mean angiogenic initiation, angiogenic growth, and overall effect that were 69%, 81%, and 94% of control values, respectively. Vincristine and 5-FU inhibited the mean overall angiogenic effect by 89% and 82% compared with control, respectively. Adriamycin inhibited overall effect 49%. Methotrexate was less effective., Conclusion: Freshly harvested breast cancer specimens develop an angiogenic response in a fibrin-thrombin clot-based angiogenesis model and respond to treatment with antineoplastic/antiangiogenic drugs. The antiangiogenic potential of commonly used breast cancer drugs varied among individual tumors. Data obtained from this model is unique and might potentially be used to further enhance the efficacy of cytotoxic regimens and individualize patient therapy.

Microarray techniques using cDNA array and comparative genomic hybridization (CGH) have been developed for several discovery applications. They are frequently applied for the prediction and diagnosis of cancer in recent years. Many studies have shown that integrating genomic data from different sources may increase the reliability of gene expression analysis results in understanding cancer progression. Therefore, developing a good prognostic model dealing simultaneously with different types of dataset is important. The challenge with these types of data is high background noise. We describe an analytical two-stage framework with a multi-parallel data analysis method named wavelet-based generalized singular value decomposition and shaving method (WGSVD-shaving). This method is proposed for de-noising and dimension-reduction during early stage prognosis modeling. We also applied a supervised gene clustering technique with penalized logistic regression with Cox-model on an integrated data. We show the accuracy of the method using a simulated dataset with a case study on Hepatocelluar Carcinoma (HCC) cDNA and CGH data. The method shows improved results from GSVD-shaving and has application in the discovery of candidate genes associated with cancer.

Background: Sentinel lymph node biopsy is an established alternative to complete lymph node dissection in some patients. We have developed a novel, radiolabeled methylene blue dye that may be a useful alternative to the traditional two-step procedure involving 99mTc-labeled colloid and unlabeled blue dye. We hypothesize that 125I-labeled methylene blue will be rapidly absorbed into the lymphatics and transported to the drainage basin containing the sentinel nodes., Materials and Methods: Rabbits footpads were injected with 1 mCi of 125I-labeled methylene blue admixed with unlabeled dye. A hand-held gamma detection device allowed tracking of radiolabeled dye to nodes in the popliteal and inguinal regions. At pre-established time points animals were sacrificed, and the nodal basin dissected. Nodal radioactivity as well as uptake of blue dye was recorded., Results: The spread of the radiolabeled methylene blue compound from the footpad to the popliteal lymph nodes occurred in 5-10 min., Conclusion: The radiolabeled dye rapidly progresses through lymphatics to the draining nodes. Use of radiolabeled methylene blue may be an attractive alternative to current two-step sentinel node techniques, as it may be less painful, and may reduce the cost associated with the time-delay between the injection of the radioactive compound and surgery., ((c) 2006 Wiley-Liss, Inc.)

Background: Previous angiogenesis models use animal tissues such as the chicken chorioallantoic membrane (CAM) or the rabbit cornea. These models may not accurately reflect the mechanisms responsible for human angiogenesis., Materials and Methods: We hypothesized that fragments of human myocardial tissue would develop an angiogenic response from the cut edges of vessels contained within the tissue. To test these hypotheses, we obtained human atrial appendage tissue at the time of cardiac bypass. Fragments of atrial tissue were then incorporated into fibrin thrombin clots. Tissue fragments were observed, and the percent of wells that developed neovessel invasion into the clot was calculated (%I). The subsequent growth of cardiac-derived microvessels was rated and scored over time (Angiogenic Index)., Results: There were 20 human atrial appendages plated (n = 24 to 60 wells/specimen) and evaluated in this model. Out of the 20, 16 (80%) atrial appendages developed an angiogenic response in the majority (>50%) of wells plated. Neovessel growth was progressive over 14 to 16 days in culture in all specimens tested. The mean angiogenic index of all specimens was 8.59 +/- 0.91., Conclusions: This human cardiac tissue-based assay might be useful to screen compounds designed for use in human trials or provide highly vascularized cardiac tissue for autotransplantation. Additionally, the assay provides the foundation to study an individual patent's cardiac tissue and its response to angiogenesis stimulators or inhibitors. This may allow the development of patient-specific therapies designed to enhance revascularization or repair of injured cardiac muscle.

Unlabelled: Some authors have suggested that chronic octreotide use enhances the efficiency of radiolabeled somatostatin receptor (sst) imaging. Conversely, desensitization of sst on tumor tissue (tachyphylaxis) may occur occasionally in patients on chronic octreotide therapy. Assuming that chronic exposure to octreotide induces tachyphylaxis, we hypothesized that chronic exposure of sst subtype 2 (sst2)-expressing cells to octreotide would downregulate binding of 111In-pentetreotide to sst and that this downregulation would be due to a reduction in the gene copy number for sst2., Methods: The clinical scenarios of acute (24 h) and chronic (2 wk) octreotide use, followed by either nuclear imaging exposure (8.6 pmol/L) or therapeutic exposure (510 pmol/L) to (111)In-pentetreotide, were modeled in vitro. Receptor binding in IMR-32 human neuroblastoma cells (high sst2 expression) and PANC-1 human pancreatic cancer cells (no detectable sst2 expression) was evaluated. Gene copy numbers for sst subtypes 1-5 in IMR-32 cells were determined by quantitative polymerase chain reaction., Results: Acute or chronic octreotide exposure at low or high doses did not significantly alter sst2 gene copy numbers or binding of either the diagnostic dose or the therapeutic dose of 111In-pentetreotide., Conclusion: In vitro exposure of cells to low or high doses of octreotide for 1-14 d does not result in the development of either tachyphylaxis or upregulation of sst as assessed by changes in gene expression or in high-affinity binding.

Targeted therapies, such as agents that inhibit angiogenesis, offer hope as complementary agents in cancer therapy. Angiogenesis-inhibiting agents have the potential for inhibiting tumor growth and limiting the dissemination of metastasis, thus keeping cancers in a static growth state for prolonged periods. Black raspberry (Rubus occidentalis) extract was discovered to be antiangiogenic (0.1% w/v) in a novel human tissue-based in vitro fibrin clot angiogenesis assay. Assay-guided fractionation of a crude black raspberry extract resulted in a highly potent antiangiogenic fraction that accounted for only 1% of the fresh weight of whole black raspberries. At 0.075% (w/v), the active fraction completely inhibited angiogenic initiation and angiogenic vessel growth. Further subfractionation of this active fraction revealed the coexistence of multiple antiangiogenic compounds, one of which has been identified as gallic acid. However, the individual subfractions did not outperform the active whole fraction. These findings suggest that an active black raspberry fraction may be a promising complementary cancer therapy. It is natural and potent enough for manageable dosing regimens. These extracts contain multiple active ingredients that may be additive or synergistic in their antiangiogenic effects. These observations warrant further investigations in animals and human trials.

Background: Angiogenesis is a critical determinant of tumor growth and the development of metastases. Tubulin inhibitors have been shown to be effective inhibitors of angiogenesis. We hypothesized that colchicine, a well-know tubulin inhibitor and 2-methoxyestradiol (2 MeOH), a novel tubulin inhibitor, would limit the initiation of a human angiogenic response and would limit subsequent neovessel growth in a dose-dependent manner., Methods: To test this hypothesis, we cultured full-thickness human placental vein discs from three placentas in a fibrin-thrombin clot model. Both colchicine and 2 MeOH were tested over a wide range of concentrations (10(-6) to 10(-12) M) to determine their effect on the percent of wells that initiated an angiogenic response (%I) and the subsequent growth (Angiogenic Index, 0-16 range) of vein-derived neovessels., Results: Colchicine at doses of 10(-6) and 10(-8) M completely inhibited the angiogenic response (CI: 95%, P < 0.0001) but lower (10(-10) to 10(-12) M) doses did not significantly inhibit angiogenesis (P = NS). Effective in vitro colchicine levels far exceed achievable non-toxic human plasma levels. In contrast, 2-methoxyestradiol decreased initiation and angiogenic growth significantly at 10(-6) M (CI: 95%, P < 0.0001), but did not significantly decrease angiogenesis at doses of 10(-8), 10(-10), or 10(-12) M. In contrast to colchicine, human plasma levels of 10(-6) M 2 MeOH are achievable clinically with little or no associated toxicity., Conclusions: Effective in vitro drug levels of 2 MeOH can be achieved in vivo, suggesting that 2 MeOH may have a role in the clinical treatment of angiogenesis-dependent diseases.

Background: Creation of protease-resistant somatostatin analogs has allowed development of these peptides as clinically useful drugs. Widespread diagnostic use of radiolabeled somatostatin analogs has enhanced interest in the binding and intracellular distribution of these peptides. The degree of drug internalization and length of drug retention may be critical for drug-induced cytotoxicity. We hypothesized that the ability of a radiolabeled peptide to bind to a cell, be internalized, and induce cytotoxicity is proportional to both the radioligand concentration and the exposure time., Materials and Methods: To test this hypothesis, somatostatin receptor-expressing cells (IMR-32) were incubated with (111)In-pentetreotide, a sst 2 preferring somatostatin analogue. Radioligand exposure time and/or concentration were varied., Results: Prolonged exposure to a fixed concentration of radioligand resulted in progressive increases in whole cell binding and internalization over time. Cells exposed to a relatively fixed number of microCi-Hr yielded constant whole cell binding and internalization. Increasing the microCi-Hr resulted in a proportionate increase in binding. Cytotoxicity was also proportional to the dose of radiation regardless of whether the exposure was internalized radiation (microCi-Hr from (111)In-pentetreotide) or from external beam radiation (cGy)., Conclusion: Both drug exposure time and drug concentration contribute to cell binding and cytotoxicity in this model and their relative contributions are inversely related.

Objective: To describe a novel in vitro human tissue-based angiogenic model that can predict an individual tumor's response to antiangiogenic drugs., Summary Background Data: A number of in vitro and in vivo angiogenesis assays exist, but they do not provide potentially useful information for the treatment of an individual patient. Clonogenic assays have been used to evaluate the response of an individual's tumor to antineoplastic agents, but these tumor fragments are cultured in an environment that does not lead to neovessel growth. The authors have previously demonstrated that human vein disks or human tumor xenograft fragments incorporated into a 0.3% fibrin-thrombin clot will develop angiogenic vessel growth from the cut edge of the vessel disk or xenograft fragment., Methods: Fresh human tumor or normal tissue disks (2 x 1 mm) from fresh surgical specimens were incorporated into fibrin-thrombin clots overlain with nutrient medium containing either 20% fetal bovine serum alone or in combination with Epothilone B, a tubulin inhibitor with antiangiogenic properties. Tissue disks were visually assessed over time to determine the percentage of wells that developed an angiogenic response. Neovessel growth, density, and length were graded at intervals using a semiquantitative visual neovessel growth-rating scheme (angiogenic index, 0-16 scale) devised in the authors' laboratory., Results: Epothilone B treatment at doses of 10-6 mol/L and 10-8 mol/L decreased the number of wells that developed an invasive angiogenic response and limited the development of vessels that invaded the matrix. At these doses, Epothilone B also caused regression of vessels in wells that had been allowed to develop an angiogenic response. Treatment of tumors or normal tissues with Epothilone B at doses less than 10-8 mol/L was ineffective., Conclusions: Epothilone B may be an effective antiangiogenic agent in a variety of tumor types. The authors speculate that this in vitro model might provide useful information to the clinician on the effect of specific antiangiogenic agents on individual tumors. This may be particularly useful in patients with tumors that, as a group, are unresponsive to treatment with antineoplastic agents.

Angiostatin, a 38-kD fragment of plasminogen, inhibits angiogenesis in both animal tumor models and in vitro endothelial cell models. However, human Angiostatin has not been tested in vitro against an intact human tissue target to determine its ability to inhibit the initiation or subsequent promotion of the human angiogenic response. We hypothesized that high doses of human Angiostatin would inhibit the development of an angiogenic response in an intact human vessel target, and would suppress the subsequent growth of blood vessels following the initiation of an angiogenic response. To test these hypotheses, full-thickness human placental vein disks were cultured for 15 days in an in vitro fibrin-thrombin clot assay. This assay system had been used to evaluate the efficacy of a wide variety of compounds. Vessels were obtained from three placentas. Treatments included a control medium plus fetal bovine serum (FBS), heparin-steroid (300 micro g/ml heparin and 350 micro g/ml hydrocortisone; a treatment known to inhibit angiogenesis) and Angiostatin at doses from 1 x 10(-4) to 1 x 10(-9) M. In the control groups, 81% of vessels initiated an angiogenic response compared to 53% of the vessels treated with heparin-steroid. Angiostatin (10(-4)-10(-9) M) decreased the initiation of an angiogenic response, but this was not statistically significant. Of the disks that initiated an angiogenic response, the mean ( +/- standard error of the mean (SEM)) semi-quantitative visual angiogenic index (AI) of the control vessels was 9 +/- 1.7 on day 15. In comparison, the mean AI of heparin-steroid treated vessels was 3.7 +/- 0.4. Angiostatin at doses of 10(-4)-10(-9) M also failed to inhibit blood vessel growth after initiation of the angiogenic response. Based on these observations, we cannot demonstrate significant activity of human Angiostatin (10(-4)-10(-9) M) against the initiation or promotion of a human angiogenic response in this in vitro model of angiogenesis using an intact human vessel target.

noni, the juice of the fruit from the Morinda citrifolia plant, has been used for centuries as a medicinal agent. We tested the effects of noni juice in a three-dimensional fibrin clot matrix model using human placental vein and human breast tumor explants as sources for angiogenic vessel development. Noni in concentrations of 5% (vol/vol) or greater was highly effective in inhibiting the initiation of new vessel sprouts from placental vein explants, compared with initiation in control explants in media supplemented with an equivalent amount of saline. These concentrations of noni were also effective in reducing the growth rate and proliferation of newly developing capillary sprouts. When used at a concentration of 10% in growth media, noni was able to induce vessel degeneration and apoptosis in wells with established capillary networks within a few days of its application. We also found that 10% noni juice in media was an effective inhibitor of capillary initiation in explants from human breast tumors. In tumor explants which did show capillary sprouting, the vessels rapidly degenerated (2-3 days) in those exposed to media supplemented with 10% noni.

Tumor growth and metastasis are dependent on the development of new blood vessels. Inhibitors of new vessel growth have been widely investigated as anti-tumor agents. Endostatin, a 20 kDa C-terminal fragment of collagen XVIII inhibits endothelial cell proliferation, induces endothelial cell apoptosis, and can both inhibit and reverse tumor growth in mice. However, human recombinant endostatin has had limited testing against human tissue targets. To investigate the effect of human endostatin on a human vessel target over a broad range of concentrations (10(-l2)-10(-4) M), human placental vein disks were grown for a period of 2 weeks in a 0.3% fibrin clot overlayed with growth medium. Disks from five individual placentas were tested. For each placenta utilized, a control (medium and 20% fetal bovine serum [FBS]) group and a group treated with heparin (300 microg/ml) and hydrocortisone 21-phosphate (350 microg/ml) (heparin-steroid) at a dose known to inhibit angiogenesis were included. Endostatin was tested at concentrations of 10(-12)-10(-4) M in medium containing 20% FBS. The rate of initiation and the angiogenic growth index (on a visually graded semi-quantitative scale of 0-16) were determined for all experimental conditions. Endostatin inhibited angiogenesis in our model only in high concentrations. At 10(-5) M, endostatin did not alter the percent of wells that initiated an angiogenic response, but significantly inhibited subsequent vessel growth. At 10(-4) M, endostatin was able to inhibit both initiation and subsequent new vessel growth. Human endostatin can inhibit the initiation of a human angiogenic response and inhibit the subsequent proliferation of human neovessels when used at high doses in a continuous exposure model.

Tumor growth and the development of metastases require an angiogenic response. Angiogenic vessels uniquely express somatostatin subtype 2 (sst 2) receptors that can transport somatostatin or its analogs into the cell. We hypothesized that radiolabeled somatostatin analogs could inhibit the angiogenic response by selectively destroying proliferating endothelial cells. We evaluated the antiangiogenic effects of 111In-pentetreotide, an sst 2-preferring somatostatin analog in a human vessel model. Disks of human placental vein were embedded in fibrin gels in culture and observed for angiogenic sprouting for 14 days. Vein disks were treated with 111In-pentetreotide (1.5, 15, and 150 microCi/mL) on the day of implantation. Control groups included disks treated with nutrient medium alone, with 111In-chloride, and with unlabeled pentetreotide. The percentage of wells that initiated an angiogenic response and the overall length and density of neovessel sprouts were assessed on Day 14. 111In-pentetreotide treatment did not completely block initiation of the angiogenic response but significantly decreased the growth of neovessels after initiation. Both the receptor-specific Auger electron-induced and nonspecific gamma radiation-mediated effects contributed to the angiotoxicity.

Introduction: Expression of somatostatin receptor subtype 2 (sst 2) in angiogenic tumor vessels appears to be homogeneous, while tumor cell expression of this receptor is often heterogeneous. We have developed a novel in vitro three-dimensional tumor angiogenesis model to study the antitumor and the antiangiogenic effects of radiolabeled somatostatin analogs. We hypothesized that targeted in situ radiation with an Auger electron-emitting radiolabeled somatostatin analog would produce receptor-specific cytotoxicity in sst 2-expressing cells., Materials and Methods: IMR-32 human neuroblastoma (sst 2-positive) and MDA MB-231 human breast cancer (sst 2-negative) xenografts were created in nude mice from monolayer cell cultures. Fragments of these tumors were embedded in three-dimensional fibrin gels supplemented with endothelial growth media and incubated for a period of 14 days. Tumor fragments were treated with 50 microCi/ml of (111)In-JIC 2DL, a sst 2-preferring somatostatin analog, or medium on Day 1. Initial angiogenic activity was determined at 48 h and the mean angiogenic score and tumoricidal responses were assessed on Day 14., Results and Conclusion: Tumoricidal effects of (111)In-JIC 2DL were seen only in sst 2-positive IMR-32 tumors. However, the angiogenic response was inhibited in both IMR-32 and MDA MB-231 tumors independent of the tumor cells' sst 2 status. Somatostatin receptor-mediated in situ radiation therapy has profound cytotoxic effects on angiogenic blood vessels and sst 2-expressing tumor cells., (Copyright 2001 Academic Press.)

Angiogenesis is a critical determinant of tumor growth and the development of metastases. Heparin, steroids, and heparin/steroid combinations have been used in a variety of in vitro models and in vivo in animal models as effective inhibitors of angiogenesis. We tested heparin, steroid and heparin/steroid combinations at a variety of concentrations to determine their effect on the human 'angiogenic switch' from a resting to a proliferative endothelium in vessels from three placentas (initiation), and the effect of these compounds on the subsequent growth of a human angiogenic response (promotion). Using full-thickness human placental vein discs cultured in three-dimensional fibrin-thrombin clots, we demonstrated that heparin (300, 3000 micrograms/ml), steroid (350, 3500 micrograms/ml), and combinations of heparin/steroid at these doses effectively blocked both initiation and promotion of a human angiogenic response in a dose-dependent fashion. We also demonstrated that high-dose steroid or heparin/steroid treatment for 15 days resulted in disruption of vessel integrity, while treatment with heparin alone produced a suppressed growth rate but had intact vessel architecture. High-dose heparin/steroid treatment could also disrupt a developed angiogenic response and retard further development of an angiogenic response following the cessation of treatment.

Unlabelled: Optimal cancer radiotherapy using Auger electron emitters requires selective localization of radionuclides in close proximity to tumor DNA., Methods: Intracellular trafficking of (125)I-Tyr1-somatostatin-14 somatotropin-release inhibiting factor (SRIF) and 2 of its analogs, (125)I-WOC 4a and (111)In-pentetreotide, was studied in human neuroblastoma cells., Results: After 24-h incubation, SRIF was degraded or recycled, whereas its protease-resistant analogs progressively accumulated in nuclear fractions. (111)In-pentetreotide binding to DNA increased over time in somatostatin receptor-positive cells but not in somatostatin receptor-negative cells., Conclusion: These in vitro studies show that prolonged exposure to radiolabeled SRIF analogs significantly increases their cellular internalization, nuclear translocation, and DNA binding. Clinically, infusion of radiolabeled somatostatin analogs may enhance tumor uptake and retention and provide more effective in situ radiotherapy.

Like many other carcinomas, prostate cancer develops resistance to inhibition by transforming growth factor (TGF)-&be;1 during oncogenesis. One proposed mechanism of TGF-&be;1 action posits action of the retinoblastoma protein (pRb) to suppress c-myc transcription to inhibit cellular proliferation. A metastatic human prostate cancer cell line, DU145, has both nonfunctional pRb and markedly reduced sensitivity to TGF-&be;1 growth inhibition. The defective rb gene in DU145 cells was replaced by a normal rb allele by microcell fusion of chromosome 13. Two subclones, DU145-Cl-I and DU145-Cl-II, were studied in vitro to determine whether the pRb restoration increased sensitivity to the inhibitory effects of TGF-&be;1. By reverse transcriptase-polymerase chain reaction, increased sensitivity to the inhibitory effects of TGF-&BE;1. By reverse transcriptase-polymerase chain reaction, parental DU145 cells had TGF-b receptors of Type I and Type II. Introduction of chromosome 13 reduced the growth rate and prolonged the G1 phase compared with the parental DU145 cell line. Moreover, responsiveness to TGF-&be;1 growth inhibition was restored in a dose-dependent manner. Transcription of c-myc was not altered by TGF-&be;1 growth inhibition. Thus, DU145 cells presumably required the presence of wildtype rb to become growth inhibited that is independent of c-myc transcription. As the entire chromosome 13 was introduced, unknown tumor suppressor genes, not only rb, may be responsible for the restoration of TGF-&be;1 growth inhibition.

Background: Radiolabeled somatostatin analogs have gained popularity for tumor imaging and have recently been used for the treatment of somatostatin receptor-expressing tumors. We have developed a novel, N-terminally extended, multiply iodinated somatostatin analog, 125I-WOC 4a, that we hypothesize will be a useful tool for the detection of and therapy for somatostatin receptor-positive tumors. To evaluate the therapeutic potential of this agent, we compared the cytotoxicity of 125I-WOC 4a in a somatostatin receptor subtype-2 (sst 2)-expressing human neurobalstoma cell line to its cytotoxicity in a somatostatin receptor-negative human pancreatic carcinoma cell line., Methods: IMR-32 neuroblastoma cells (sst 2-positive) and PANC-1 human pancreatic cells (sst 2-negative) were incubated with 125I-WOC 4a at doses ranging from 0.1-100 CPM/cell for 48 h and cell viability was assessed by a colorimetric (MTT) cell viability assay. Subsequently, IMR-32 cells were incubated with either control medium, 125I-WOC 4a (1 cpm/cell) alone, 125I-WOC 4a with 10(-6) M octreotide acetate, 125I (1 cpm/cell) alone, 125I with octreotide acetate, or octreotide acetate alone for 48 h, washed, and cryopreserved for 4 weeks. Cells were then thawed, replated, and allowed to acclimate for 48 h. Cell viability was assessed by trypan blue exclusion and a colorimetric assay., Results: Following short-term exposure, 125I-WOC 4a induced dose-dependent cytotoxicity in IMR-32 cells (P < 0.05 by ANOVA), but not in the PANC-1 cells. After exposure to 125I-WOC 4a (1 cpm/cell) for 48 h followed by a 4-week cryopreserved exposure, significant cytotoxicity was induced in IMR-32 cells (P < 0.05 by ANOVA) which was not seen in cells treated with 125I alone or 125I with 10(-6) M octreotide acetate. Simultaneous exposure to 125I-WOC 4a and octreotide acetate was also cytotoxic., Conclusion: 125I-WOC 4a induces receptor-specific cytotoxicity following both short- and long-term drug exposures. This radiopharmaceutical may be useful for localizing or treating somatostatin receptor-positive tumors.

Prostate cancer eventually becomes androgen resistant, resumes growth, and kills the patient. Characterization of genetic events that lead to androgen refractory prostatic neoplasia has revealed the frequent overexpression of c-myc and uncontrolled prostate cancer proliferation. A novel strategy to combat advanced prostate cancer utilized a replication incompetent retrovirus that contained the mouse mammary tumor virus (MMTV) promoter within the retroviral vector to allow transcription of antisense c-myc gene within target prostate tumor cells. The transduction of cultured DU145 cells by XM6:MMTV-antisense c-myc RNA retrovirus did not affect cell proliferation in culture, yet a single direct injection of MMTV-antisense c-myc viral media into established DU145 tumors in nude mice produced a 94.5% reduction in tumor size compared to tumors treated with control virus MTMV sense fos and untreated tumor by 70 days. Two animals in the antisense c-myc-treated group had complete regression of their tumors. Histopathological examination of the tumors revealed that MMTV-antisense c-myc-transduced DU145 tumors had increased tumor cell differentiation, decreased invasion, and a marked stromal response. The mechanism for the antitumor effect of MMTV-antisense c-myc retrovirus appears to be suppression of c-myc mRNA and protein, and decreased bcl-2 protein. The in vivo transduction of prostate cancer cells with MMTV-antisense c-myc retroviruses reduced tumor growth by suppressing c-myc, resulting in the down-regulation of bcl-2 protein. Consequently, the MMTV-antisense c-myc retrovirus may be useful for gene therapy against advanced, hormone-refractory prostate cancer.

Background: Prostate cancer eventually becomes androgen-independent, suggesting that growth factors such as TGF beta 1-3 may potentially contribute to prostate neoplasia. The pattern and level of TGF beta 1-3 protein expression in normal and malignant human prostate are unknown., Methods: An immunohistochemical study was undertaken to analyze TGF beta 1, TGF beta 2, and TGF beta 3 protein in malignant and adjacent normal prostates from 25 patients who had clinically localized prostate cancer., Results: Normal prostate exhibited similar TGF beta 1 immunostaining in stromal and epithelial cells, whereas TGF beta 2 and TGF beta 3 protein staining was greater in the epithelial relative to the stromal compartments. In malignancy, prostate epithelial cells had higher TGF beta 1 and TGF beta 2 immunostaining than either the surrounding stromal cells or their normal prostatic epithelial counterparts. Although TGF beta 3 staining intensity was similar for both malignant and normal prostate epithelial cells, the pattern of staining switched from uniform apical to diffuse protein staining in malignant prostate glands., Conclusions: Prostate cancer was associated with alterations of TGF beta 1, TGF beta 2, and TGF beta 3 expression by prostatic epithelial cells which may play a role in prostatic carcinogenesis.

Objectives: Tumor biomarkers to detect prostate cancer earlier may reduce prostate cancer deaths. Transforming growth factor-beta1 and -beta2 (TGF-beta1 and -beta2) become overexpressed in prostate cancer and might be useful tumor markers of prostate cancer., Methods: Plasma and urinary TGF-beta1 and plasma TGF-beta2 levels were studied preoperatively in 74 consecutive patients who had prostate cancer and underwent radical prostatectomy and were compared with those of 29 similarly aged male control patients who had no clinical evidence of prostate cancer., Results: Plasma TGF-beta1 levels were similar in both prostate cancer and control groups and did not correlate with serum prostate-specific antigen (PSA), clinical and pathologic stages, or Gleason grade. Urinary TGF-beta1 levels, however, increased 3.5-fold in patients with prostate cancer relative to controls and tended to be higher with advancing clinical and pathologic stages. Plasma TGF-beta2 levels, like plasma TGF-beta1 levels, were similar for both the study and control groups, but when stratified by pathologic stage or Gleason grade, patients with prostate cancer with pathologic Stage T2a and Gleason grade of 3 or less had significantly increased plasma TGF-beta2 levels as compared with either control patients or patients with prostate cancer with pathologic Stages T2b/T2c and T3/T4 or Gleason grade of 4 or more, suggesting that early prostate cancer may contribute to plasma TGF-beta2 levels., Conclusions: Unlike plasma TGF-beta1 levels, urinary TGF-beta1 and plasma TGF-beta2 levels were higher in patients with prostate cancer and may be useful biomarkers of prostate cancer.

It is current practice in many clinical trials evaluating new chemotherapy regimens for the treatment of advanced prostate cancer to use prostate-specific antigen (PSA) decline as a response criteria with the assumption that the level of PSA reflects the efficacy of chemotherapy. Advanced prostate cancer is heterogeneous; therefore, the validity of PSA decline as a measurable end point was studied in advanced human prostate-cancer cell lines: androgen-sensitive LNCaP and androgen-insensitive PC3 cells. Each cell line was grown for 4 days with escalating doses of Adriamycin or vinblastine. Cell counts, intracellular PSA concentrations, and secreted PSA levels were determined daily for 4 days. Untreated LNCaP cells had constant secretion of PSA per cell. In contrast, LNCaP cells treated with Adriamycin or vinblastine had an 80% reduction in cell numbers and a 3-fold increase in secreted PSA per cell by day 4. In contrast, PC3 cells had a different response to Adriamycin and vinblastine. Both drugs reduced cell numbers by 97% of control values and suppressed PSA production in the remaining viable cells by 4 days in culture. Thus, prostate-cancer cell production of PSA is variable with chemotherapy and the PSA level may not accurately reflect the actual tumor response to chemotherapy.

The observation that advanced prostate cancer has reduced sensitivity to transforming growth factor βI (TGFβI) growth inhibition suggests that the acquisition of TGFβI resistance may play a role in prostate tumor progression. Using the Dunning R3327 rat prostate adenocarcinoma model, it was determined that prostate carcinoma cells became less responsive to TGFβI growth inhibition with differentiated tumors more resistant to TGFβI. A TGFβ receptor defect was not found in advanced prostate carcinoma cells because both the type I and II TGFβ receptors were present and functional. Moreover, TGFα/epidermal growth factor receptor (EGFR) and basic fibroblast growth factor (bFGF)/fibroblast growth factor receptor (FGFR) autocrine stimulatory pathways, which may potentially counter-regulate the inhibitory effects of TGFβI, were not present with prostate cancer progression. However, the likelihood that other prereceptor stimulatory pathways or TGFβ postreceptor signaling alterations are responsible for reduced sensitivity to TGFβI growth inhibition remains to be elucidated.

Bone morphogenetic proteins (BMPs) have multiple biologic functions, including bone formation and embryonic induction. One of these proteins, BMP-6, was reportedly expressed at high levels in human prostate cancers that had also metastasized to bone. This study investigated both BMP-6 mRNA and protein expression in normal and malignant rat and human prostate tissues. BMP-6 was detected in both rat normal prostate and in Dunning rat-prostate adenocarcinoma sublines. The levels of BMP-6 mRNA and protein were similar for normal and malignant rat prostate, regardless of the metastatic potential. Moreover, castration had no apparent effect on BMP-6 production in rat normal ventral prostate, suggesting an androgen-independent gene regulation of this protein. BMP-6 mRNA and protein were also produced by normal and neoplastic human prostate cancer (radical prostatectomy specimens and human carcinoma cell lines DU145 and PC3). BMP-6 mRNA and protein expression, however, was higher in prostate cancer as compared with adjacent normal prostate, with higher-grade tumors (Gleason score of 6 or more) having greater BMP-6 immunostaining than the lower-grade tumors (Gleason score of 4 or less). Taken together, these results suggest that BMP-6 protein expression may serve as a potential marker for prostate cancer but not as a metastatic marker. Moreover, BMP-6 may contribute to prostate neoplastic behavior even in the absence of androgens.

Peritubular cells that surround the seminiferous tubules have been shown to produce a paracrine factor, termed P-Mod-S, that has dramatic effects on Sertoli cell function in vitro and is postulated to be important in the control of testicular function. The current study was designed to determine whether P-Mod-S has the ability to regulate Sertoli cell function during pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats which correspond to the prepubertal, mid-pubertal, and late pubertal stages of development. Histochemical analysis of cultured cells isolated from each age group was performed to establish the purity of the cell populations used. Testicular transferrin production by Sertoli cells was used as a marker of cellular differentiation. Basal production of transferrin by the cultured cells was found to increase during the pubertal period. P-Mod-S stimulated transferrin production by Sertoli cells isolated from 10-, 20-, and 35-day-old rats. FSH appears to enhance the ability of Sertoli cells to respond to P-Mod-S with cells obtained from 10-day-old rats. Sertoli cells from 35-day-old rats were nonresponsive to regulatory agents such as FSH. P-Mod-S alone, however, significantly stimulated transferrin production by Sertoli cells from this more adult stage of development. P-Mod-S was the only individual regulatory agent tested that could stimulate transferrin production by Sertoli cells from 35-day-old rats. Results indicate that P-Mod-S has the ability to regulate Sertoli cell function throughout pubertal development. Observations suggest that P-Mod-S and FSH may act together in the prepubertal testis to promote Sertoli cell differentiation and that P-Mod-S may act in the adult testis to maintain optimal Sertoli cell function and differentiation.

Regulatory interactions have been shown to occur between all the testicular cell types considered. The paracrine factors mediating these interactions generally influence either cellular growth or differentiation. The regulation of cellular growth is essential in the developing testis and is required for the maintenance of spermatogenesis in the adult testis. The rapid rate of germinal cell proliferation and the continuous but slowed growth of the peritubular cells and Leydig cells requires the presence of specific growth factors in the adult. Therefore, cell-cell interactions have evolved that involve growth factors such as IGF, TGF-alpha, TGF-beta and NGF. Other growth factors such as FGF or less characterized components like the seminiferous growth factor (SGF) also may be involved in the paracrine regulation of testis cell growth. An alternate cellular parameter to cell growth to consider is the regulation of cellular function and differentiation. A number of endocrine agents and locally produced paracrine factors have been shown to control and maintain testis cell function and differentiation. Cell-cell interactions mediated by factors such as androgens, POMC peptides, and PModS are all primarily directed at the regulation of cellular differentiation. Therefore, the agents which mediate cell-cell interactions in the testis can generally be categorized into factors that regulate cell growth or those which influence cellular differentiation. The specific cell-cell interactions identified will likely be the first of a large number of cellular interactions yet to be investigated. Although a number of potentially important cell-cell interactions have been identified, future research will require the elucidation of the in vivo physiological significance of these interactions. The existence of different cell types and potential cell-cell interactions in a tissue implies that the actions of an endocrine agent on a tissue will not simply involve a single hormone and single cell. The endocrine regulation of testis function will have effects on cell-cell interactions and be affected by local cell-cell interactions. The ability of LH to influence Leydig cell androgen production promotes a cascade of interactions mediated through several cell types to maintain the process of spermatogenesis. FSH actions on Sertoli cells also promote cell-cell interactions that influence germinal cell development, peritubular myoid cell differentiation and Leydig cell function. Therefore, elucidation of the endocrine regulation of testis function requires an understanding of the local cell-cell interactions in the testis.

Sertoli cells were isolated and cultured in the absence or presence of extracellular matrix (ECM) to determine whether ECM may influence Sertoli cell function on a molecular level. As previously described, a morphological analysis of the cells indicated that ECM allows the expression of a columnar histotype and the formation of junctional complexes. The combined actions of ECM and hormones were found to have a profound effect in promoting the expression of a polarized Sertoli cell morphology. In our investigation of the effects of ECM on Sertoli cells, we used transferrin and androgen-binding protein (ABP) production as biochemical markers of Sertoli cell function. The presence of ECM was found to cause a 25% increase in the basal level of transferrin production; however, ECM had no effect on the basal level of ABP production by Sertoli cells. Regulatory agents such as follicle-stimulating hormone (FSH) and a combination of FSH, insulin, retinol, and testosterone stimulated the production of both transferrin and ABP. The ability of hormones to stimulate these Sertoli cell functions was not influenced by the presence of ECM. Similar results were obtained with 2-microns- or 50-microns-thick ECM and with a seminiferous tubule biomatrix preparation. ECM was found to increase the maintenance of long-term Sertoli cell cultures; however, the decline in Sertoli cell functional integrity, which occurs during cell culture, was not affected by the presence of ECM. An additional functional parameter examined was the radiolabeled proteins secreted by Sertoli cells. ECM did not promote the production or affect the electrophoretic profile of Sertoli cell-secreted proteins under basal or hormonally stimulated conditions. Combined results indicated that although ECM allowed the expression of a normal Sertoli cell histotype, ECM had no major effects on the Sertoli cell functions analyzed nor on the hormonal regulation of these functions. The inability of ECM to affect Sertoli cell function on a molecular level is discussed with regard to environmental as opposed to regulatory cellular interactions. Our observations imply that dramatic effects of ECM on cell morphology do not necessarily correlate to subsequent effects on cellular function.

The cellular location of fibronectin expression within the seminiferous tubule was investigated in order to better understand testicular cell functions and cell-cell interactions. Peritubular cells were shown to actively synthesize and secrete fibronectin in culture by the detection of a radiolabeled 220 kDa secreted protein that is immunologically similar to fibronectin and by the quantitation of fibronectin in peritubular cell conditioned medium with a fibronectin enzyme-linked immunosorbent assay. Sertoli cells did not produce detectable levels of fibronectin when assayed by either of these procedures. A 6.5 kb fibronectin messenger RNA was detected in freshly isolated or cultured peritubular cells, but no fibronectin gene expression was detected in Sertoli cells or developing germinal cells. Combined results imply that the peritubular cells are the only apparent site of fibronectin expression within the seminiferous tubule. During the development of the testis the levels of fibronectin expression increased to a maximum at early puberty (15-day-old rats) and then slowly declined. The results demonstrate that fibronectin can be utilized as a unique functional and biochemical marker for peritubular cells when compared to other cell types in the seminiferous tubule. Production of fibronectin by peritubular cells provides an example of the ability of peritubular cells and Sertoli cells to cooperate in the production of individual components of the basement membrane of the seminiferous tubule. This cellular interaction is an example of a mesenchymal/stromal-epithelial interaction which is postulated to be important for the physiology of many tissues.

The regulation of Sertoli cell function was investigated through an examination of the effects of various hormones, regulatory agents, and culture conditions on testicular transferrin and androgen-binding protein (ABP) synthesis and steady state levels of mRNA. FSH stimulated both transferrin and ABP production 2-fold above control levels. Interestingly, FSH had a differential effect on transferrin and ABP mRNA levels, with 1.25- and 2.0-fold respective increases in steady state levels of mRNA. Insulin and retinol stimulated both transferrin and ABP synthesis in a similar manner. Testosterone had no significant effect on either transferrin or ABP mRNA levels or synthesis. Maximum stimulation of both transferrin and ABP production occurred when Sertoli cell cultures were treated with a combination of FSH, insulin, and retinol, which resulted in a greater than 4-fold stimulation of synthesis and 2-fold stimulation of gene expression. Optimal transferrin and ABP secretion occurred between days 4-6 of Sertoli cell culture and subsequently declined. Sertoli cell number decreased with time in culture, such that approximately a 50% loss of cells was observed after 10 days of culture. The responsiveness of Sertoli cells to regulatory agents was altered by cell density, with a maximum responsiveness achieved at a density of 12 micrograms DNA/2 cm2 for both transferrin and ABP. As the cell density deviated from this level the responsiveness of cells to regulatory agents decreased and approached control values. These observations indicate that the culture conditions and the method of data normalization are important parameters in an analysis of the hormonal regulation of Sertoli cell function. FSH actions on Sertoli cells increased both cellular and excreted cAMP levels but had no effect on cGMP levels. (Bu)2 cAMP affected transferrin and ABP mRNA levels and synthesis in a similar manner, with approximately a 3-fold increase in synthesis and a 1.5-fold increase in steady state levels of mRNA. The minimum and maximum effective concentrations of (Bu)2AMP for both proteins were 1 and 10 microM, respectively. Observations imply that regulatory agents that act via a cAMP-mediated signal transduction mechanism, such as FSH, will probably have similar actions on transferrin and ABP production. In addition, data obtained with insulin and retinol indicate that transferrin and ABP production can be similarly regulated with cAMP-independent signal transduction mechanisms. Results indicate that transferrin and ABP mRNA levels and synthesis are regulated in a coordinate manner with the regulatory agents and culture conditions evaluated.(ABSTRACT TRUNCATED AT 400 WORDS)

The hypothesized relationship between androgen-binding protein (ABP) and sperm maturation was investigated using a mutant rodent: the restricted rat. The seminiferous epithelium of these animals undergoes a spontaneous degeneration, but changes are progressive. Restricted rats in the transition to infertility were used to determine if changes in ABP were related to the decreased fertility found in these animals. Fertilizing ability was determined by insemination of cauda epididymal spermatozoa into hormonally primed female rats and examination of ova for evidence of fertilization 48 h later. Epididymal and testicular tissues were analyzed for ABP using a charcoal assay. Androgen levels were determined by RIA. Testicular weights were significantly reduced compared to those of normal littermates in restricted rats at all ages; epididymal weights were significantly reduced in rats 140 days and older. Among restricted rats, sperm fertilizing ability was variable, but was significantly lower than that in normal littermates; it was consistently highest at 90 days of age. Epididymal ABP content (picomoles per organ) was significantly reduced in restricted rats at all ages; peak values occurred at 90 days. Testicular ABP content was significantly reduced only in the youngest and oldest animals. Plasma testosterone levels were not statistically lower than those found in normal littermates, and ventral prostate weights were maintained at normal levels in all four groups of animals. A significant positive correlation existed between sperm fertilizing ability and epididymal ABP, but not between sperm fertilizing ability and plasma testosterone. Since ABP is an index of Sertoli cell function, these data indicate that sperm fertilizing ability is closely related to Sertoli cell function and/or ABP.

A microassay for the androgen receptor was developed to investigate the cellular distribution of receptor in freshly isolated testicular cell types. The microassay uses an androgen affinity ligand, 17 beta-dihydrotestosterone bromoacetate. Binding of this ligand by the androgen receptor is rapid and irreversible, which permits the development of a highly sensitive assay. The androgen receptor microassay is completed within 4 h and detects receptor in as little as 0.5 micrograms cellular protein. There was no detectable binding of the affinity label by albumin or Sertoli cell-secreted proteins, including androgen-binding protein. Androgen receptor was found in cellular sonicates of human foreskin fibroblast, rat ventral prostate, rat kidney, and rat liver. Although the relative distribution of receptor was similar to that obtained using a traditional equilibrium binding assay, the levels of receptor were significantly higher using the microassay. The androgen receptor microassay was subsequently used to investigate the receptor in isolated testicular cell types. Androgen receptor was detected in freshly isolated peritubular myoid cells (80 fmol/micrograms DNA), Sertoli cells (88 fmol/micrograms DNA), and Leydig cells (35 fmol/micrograms DNA). No androgen receptor was detected in a mixed population of germ cells. Hormones were not found to influence androgen receptor levels in cultured peritubular cells or Sertoli cells. Electrophoretic analysis of androgen receptor radiolabeled with the affinity ligand demonstrates a single 52-kDa form of the receptor in peritubular cells, Sertoli cells, and Leydig cells. The size of the androgen receptor species detected in the rat testicular cell types was slightly smaller than the 56-kDa protein detected in a human fibroblast cell line. The current study demonstrates the utility of the microassay and affinity ligand to investigate androgen receptor biology. Data indicate that androgen receptors are present in several testicular cell types and suggest that the control of testicular function by androgens probably involves actions on multiple cell types.

Hypophysectomized rats maintained with 2 mg pregnenolone have low plasma testosterone levels, but rete testis levels are normal. This model system was used to examine the importance of androgen-binding protein (ABP) in maintaining high luminal androgen concentrations for the development and maintenance of sperm fertilizing ability in the epididymis. Hypophysectomized rats were injected with pregnenolone (1, 0.5, 0.2, or 0.02 mg/100 g BW) for 14 days, starting 1 day after surgery. Sham-operated rats and a group of hypophysectomized rats were injected with oils as controls. At the end of the experimental period, sperm fertilizing ability, tissue weights, ABP, and testosterone levels were determined. The 1- and 0.5-mg doses of pregnenolone resulted in rete testis testosterone levels that were 67% and 29%, respectively, of the levels found in sham-operated animals. Plasma testosterone levels were not different from levels found in hypophysectomized, oil-injected controls with any of the pregnenolone doses. The three highest doses of pregnenolone resulted in levels of testicular ABP that were not statistically different from sham-operated levels (2 pmol/organ). Epididymal ABP content was maintained at sham-operated control levels (30 pmol/organ) with the 1-mg dose; with the 0.5- and 0.2-mg doses, ABP levels were 76% and 73% of sham-operated levels, respectively. Epididymal ABP specific content (picomoles per 100 mg tissue) was maintained at sham-operated control levels with the 1-, 0.5-, and 0.2-mg doses. Sperm fertilizing ability was maintained at sham-operated control levels with the three highest doses of pregnenolone. The 0.02-mg dose of pregnenolone resulted in values that were not statistically different from hypophysectomized, oil-injected control values for all parameters tested. A significant positive correlation existed between ABP and sperm fertilizing ability. These data demonstrate that testicular and epididymal ABP levels can be maintained in hypophysectomized rats with pregnenolone treatment alone, and that sperm fertilizing ability can be maintained when intraluminal androgen levels are low and ABP levels are near normal. This suggests that ABP may be important in the maintenance of normal sperm fertilizing ability.

A testicular paracrine factor, P-Mod-S, was purified from conditioned medium obtained from serum-free cultures of peritubular cells. Stimulation of testicular transferrin production by cultured Sertoli cells was utilized as a bio-assay for P-Mod-S. A bioactive protein with an apparent molecular weight of 50,000 under physiological conditions was isolated by high pressure size exclusion chromatography. P-Mod-S was found to have an affinity for heparin and bound to a heparin affinity column. Two forms of P-Mod-S were purified with reverse-phase chromatography. The less hydrophobic form was referred to as P-Mod-S (A) and is a 56,000 molecular weight protein. The more hydrophobic form was referred to as P-Mod-S (B) and is a 59,000 molecular weight protein. Purification of P-Mod-S (A) and P-Mod-S (B) from peritubular cell-radiolabeled secreted proteins revealed that both proteins contain radioactivity. This result demonstrates active synthesis and secretion of P-Mod-S by peritubular cells. Although the amino acid composition of the two proteins indicates distinct differences in the content of several amino acids, the relationship of P-Mod-S (A) and P-Mod-S (B) is unknown at present. A greater than 1000-fold increase in the specific activity of P-Mod-S was achieved with the purification procedure utilized. P-Mod-S can account for essentially all the bioactivity present in crude peritubular cell-secreted protein preparations. The effects of the two forms of P-Mod-S on both transferrin and androgen-binding protein production by Sertoli cells was examined. Purified forms of P-Mod-S were found to have a greater effect on Sertoli cell function than any individual regulatory agent previously known to influence the cell, including follicle-stimulating hormone. The significance of peritubular cell-Sertoli cell interactions mediated via P-Mod-S to spermatogenesis and testicular function is discussed, as well as insight provided into general mesenchymal-epithelial cell interactions.

Testicular peritubular myoid cells secrete a paracrine factor that is a potent modulator of Sertoli cell functions involved in the maintenance of spermatogenesis. These cells also play an integral role in maintaining the structural integrity of the seminiferous tubule. To better understand this important testicular cell type, studies were initiated to characterize cultured peritubular cells using biochemical and histochemical techniques. The electrophoretic pattern of radiolabeled secreted proteins was similar for primary and subcultured peritubular cells and was unique from that of Sertoli cells. Morphologic differences between Sertoli cells and peritubular cells were noted and extended with histochemical staining techniques. Desmin cytoskeletal filaments were demonstrated immunocytochemically in peritubular cells, both in culture and in tissue sections, but were not detected in Sertoli cells. Desmin is proposed to be a marker for peritubular cell differentiation as well as a marker for peritubular cell contamination in Sertoli cell cultures. Peritubular cells and Sertoli cells were also stained histochemically for the presence of alkaline phosphatase. Staining for the alkaline phosphatase enzyme was associated with peritubular cells but not with Sertoli cells. Alkaline phosphatase is therefore an additional histochemical marker for peritubular cells. Biochemical characterization of peritubular cells relied on cell-specific enzymatic activities. Creatine phosphokinase activity, a marker for contractile cells, was found to be associated with peritubular cells, while negligible activity was associated with Sertoli cells. Alkaline phosphatase activity assayed spectrophotometrically was found to be a useful biochemical marker for peritubular cell function and was utilized to determine the responsiveness of primary and subcultured cells to regulatory agents. Testosterone stimulated alkaline phosphatase activity associated with primary cultures of peritubular cells, thus supporting the observation that peritubular cells provide a site of androgen action in the testis. Retinol increased alkaline phosphatase activity in subcultured peritubular cells. Alkaline phosphatase activity increased in response to dibutyryl cyclic adenosine monophosphate (AMP) in both primary and subcultured peritubular cell cultures. Observations indicate that the ability of androgens and retinoids to regulate testicular function may be mediated, in part, through their effects on peritubular cells. This provides additional support for the proposal that the mesenchymal-epithelial cell interactions between peritubular cells and Sertoli cells are important for the maintenance and control of testicular function. Results imply that the endocrine regulation of tissue function may be mediated in part through alterations in mesenchymal-epithelial cell interactions.