The effect of imatinib mesylate on the proliferation, invasive ability, and radiosensitivity of retinoblastoma cell lines.

PURPOSE: Our aim was to evaluate the potential effect of imatinib mesylate (IM), a small molecule that specifically inhibits the tyrosine quinase receptors, on the proliferation and invasive abilities of two human retinoblastoma (Rb) cell lines. Furthermore, the ability of IM to radiosensitize Rb cells was evaluated. The potential targets of IM (C-kit, PDGRF-α and -ß, and c-Abl) were also investigated in these cell lines. METHODS: Two human Rb cell lines (WERI-RB-1 and Y79) were cultured under normal growth conditions. An MTT-based proliferation assay and a Matrigel invasion assay were performed with and without exposure to 10 µM of IM. The cells were also irradiated with graded dosages of 0, 2, 4, 6, 8, and 10 Gy with and without IM and their proliferations rates were analyzed. Western blot and immunocytochemical analysis of cytospins were performed to evaluate the expression of C-kit, PDGRF-α and -ß, and c-Abl. RESULTS: When IM was added to both cell lines a statistically significant (P<0.05) reduction in proliferation and invasive ability were observed. Exposure to IM also significantly increased the radiosensitivity of both Rb cell lines. The c-Abl expression was strongly positive, PDGRF-α and -ß expression were also positive but the C-kit expression was negative in both cell lines. CONCLUSIONS: These results indicate that Gleevec may be useful as an adjuvant treatment in Rb patients, specially those considered for radiation therapy.

Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis.

The liver is a major site of metastasis for human malignancies, yet the factors that regulate tumor cell survival and growth in this organ remain elusive. Previously, we reported that M-27(IGF-IR) murine lung carcinoma cells with ectopic insulin-like growth factor-1 (IGF-I) receptor overexpression acquired a site-specific, liver-metastasizing potential. Gene expression profiling and subsequent RNA and protein analyses revealed that this was associated with major changes to the expression of extracellular matrix (ECM) protein-encoding genes including type III, IV and XVIII collagen genes, and these changes were also observed in the respective tumors in vivo. Because type IV collagen was the most prominently altered ECM protein in this model, we further analyzed its functional relevance to liver metastasis. M-27 cells stably overexpressing type IV collagen α1 and α2 chains were generated and their growth and metastatic properties investigated. We found that these cells acquired a site-selective growth advantage in the liver and this was associated with cell rescue from anoikis in a collagen IV/α2 integrin/FAK-dependent manner and increased responsiveness to IGF-I. Conversely, collagen IV or focal adhesion kinase (FAK) silencing by small-interfering RNA in highly metastatic tumor cells enhanced anoikis and decreased liver metastases formation. Moreover, analysis of human surgical specimens revealed uniformly high collagen IV expression in 65/65 hepatic metastases analyzed, regardless of tissue of origin, whereas it was variable and generally low in 50/50 primary colorectal carcinoma specimens examined. The results suggest that collagen IV-conveyed signals are essential cues for liver metastasis in diverse tumor types and identify mediators of collagen IV signaling as potential therapeutic targets in the management of hepatic metastases.

Expression of the metastasis suppressor gene KISS1 in uveal melanoma.

PURPOSE: Uveal melanoma (UM) is the most common primary malignant intraocular tumour in adults. Forty-five percent of UM patients develop metastasis within 15 years of initial diagnosis. KISS1, a human metastasis suppressor gene, has been reported to play a role in various human malignancies. The purpose of this study was to investigate the expression of KISS1 in UM and its potential value as a prognostic marker. METHODS: Thirty-seven cases of paraffin-embedded human UM specimens were immunostained with a KISS1 antibody. Clinical-pathological data were obtained. The relationship between the clinical-pathological data and the expression of KISS1 was evaluated. Moreover, the survival rates of the patients were also assessed. Five UM cell lines (92.1, OCM-1, MKTBR, UW1 and SP6.5) were assayed for KISS1 expression. In addition, real-time PCR was used to determine mRNA levels of KISS1and its receptor GPR54in these cell lines. RESULTS: The immunohistochemical results of KISS1 expression displayed cytoplasmic staining in 84% of UM specimens. Low KISS1 expression was associated with a higher risk of metastatic disease (P<0.05). Furthermore, we found that KISS1 was expressed in all five UM cells lines. Real-time PCR analysis confirmed the presence of both KISS1and its receptor GPR54in all five human UM cell lines. CONCLUSIONS: To the best of our knowledge, this is the first time that KISS1has been characterized in UM. The correlation between KISS1 expression and UM survival rate suggests an important role for KISS1as a prognostic marker in this particular tumour.

Identification of circulating malignant cells and its correlation with prognostic factors and treatment in uveal melanoma. A prospective longitudinal study.

PURPOSE: Most uveal melanoma patients (UMP) do not show evidence of metastases upon diagnosis. However, despite local tumour control, approximately 50% of them will develop metastases. These findings suggest that malignant cells may have already disseminated by the time of initial diagnosis. The purpose of the study was to detect circulating malignant cells (CMCs) in UMP and to correlate them with prognostic factors and therapy. METHODS: Nested reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect CMCs. In each UMP, blood was collected every 3 months. In each visit, 20 RT-PCR tests were performed. The date of diagnosis, largest tumour dimension, type, and date of treatment were obtained. RESULTS: A total of 30 UMP were enrolled. Five patients were enrolled at the time of diagnosis and 25 patients between 1 and 17 years following diagnosis. No UMP showed clinical evidence of metastasis. A total of 136 visits were registered, 1360 samples collected, and 2720 RT-PCRs performed. CMCs were identified in 29 patients in 119 visits (87.5%). However, in each visit, a low number of positive tests were recorded. CMCs were found in newly diagnosed, irradiated, enucleated, and observed patients regardless of tumour size and time period following treatment. CONCLUSIONS: Uveal melanoma (UM) is not a localized ocular disease. CMCs were recorded at initial diagnosis confirming the early metastatic nature of UM. CMCs were present following treatment, including enucleation, demonstrating that CMCs are capable of disseminating and surviving, possibly as micrometastasis, which would contribute to the pool of CMCs at a later stage. Systemic therapy should be evaluated.

Role of interferon-gamma in the priming of decidual macrophages for nitric oxide production and early pregnancy loss.

We have previously shown that both priming and triggering signals were needed for nitric oxide production by decidual macrophages and that nitric oxide was responsible for embryo wastage. In this study, we investigated the role of IFN-gamma as the primary signal for macrophage activation in early embryo loss. IFN-gamma-deficient (GKO) and heterozygous F1 control mice were injected with lipopolysaccharide (LPS) at day 7 of gestation. The results showed that the GKO mice were more resistant to LPS-induced embryo loss than the wild type. This suggested that IFN-gamma was needed for LPS-induced embryo resorption and that decidual macrophages from pregnant GKO mice were not primed and could not be activated when given LPS. Further, the results showed that IFN-gamma mRNA was simultaneously expressed in the same embryos that also expressed mRNA markers for macrophage activation (TNF-alpha and iNOS), indicating that macrophage activation could be a consequence of IFN-gamma production. Similarly, we investigated the role of IL-12 as a switch cytokine capable of eliciting TH1-associated cytokine production including IFN-gamma. The results showed that IL-12 mRNA expression was correlated with IFN-gamma expression and macrophage activation. In this in vivo study, we showed for the first time that spontaneously increased decidual IFN-gamma expression is detrimental to embryo survival.

Immunohistochemical expression of S-100 beta in choroidal melanomas.

OBJECTIVE: To investigate the expressivity of S-100 beta antibodies in choroidal melanomas and to compare it with that of S-100 protein and HMB-45. DESIGN: Twenty-seven choroidal melanoma specimens obtained from the McGill University Ophthalmic Pathology Registry were classified as spindle cell, epithelioid cell or mixed-cell type. Immunohistochemistry was performed using the standard peroxidase-antiperoxidase technique with monoclonal HMB-45, polyclonal S-100, polyclonal S-100 beta and monoclonal S-100 alpha beta antibodies in formalin-fixed, paraffin-embedded sections. OUTCOME MEASURE: Intensity of immunoreaction. The result was considered positive when at least five focal areas of stained cells were observed within the tumour. RESULTS: All 27 tumours were positive for HMB-45, 19 (70%) for S-100, 23 (85%) for S-100 beta, and 21 (78%) for S-100 alpha beta. No correlation was found between the intensity of the immunoreaction and cell classification. CONCLUSIONS: HMB-45 is the most reliable marker for choroidal melanomas. S-100 beta is a more sensitive marker than S-100 for choroidal melanomas regardless of cell type. Contrary to previous reports, S-100 beta should not be considered a useful immunomarker to differentiate between primary choroidal melanoma and cutaneous melanoma metastatic to the choroid.

Decidual infiltration and activation of macrophages leads to early embryo loss.

PROBLEM: There is considerable controversy concerning the root cause and mechanisms of early embryo loss. It has been suggested that most pregnancy losses occur due to morphogenetic anomalies of the embryo. It has also been suggested that the maternal specific immune system rejects the embryo. METHODS: Existing data on the cell and molecular biology of early embryo loss in murine experimental models is reviewed. RESULTS: Using the CBA(female) x DBA/2(male) model of early embryo loss, it has been established that maternal inflammatory cells infiltrate the decidua basalis of all implantation sites within 48 hr after implantation. For most embryos, the relatively low numbers of macrophages (Mphi) and natural killer-like (NK-like) cells of maternal origin remain relatively constant after day 8, whereas 20-30% of the embryos show a significant increase in inflammatory cells in the maternal decidua, corresponding to the incidence of early embryo resorption visible at day 12. Evidence will be reviewed to suggest that decidual NK-like cells are not cytolytic but may be producing the Mphi-activating cytokine interferon gamma (IFN-gamma), which activates decidual Mphi and other cells. Furthermore, embryo loss is ameliorated by in vivo treatment with anti-IFNgamma or anti-NK antisera, indicating that NK-like cells and/or IFNgamma are required for embryo loss, but not for embryo survival. In resorbing embryos, the inflammatory Mphi show evidence of having been primed during early pregnancy, in that in vitro incubation with lipopolysaccharide induced the production of tumor necrosis factor alpha and nitric oxide. CONCLUSION: These findings support the concept that early embryo loss is a nonspecific event mediated by the triggering of cytotoxin production by primed decidual macrophages.

Nitric oxide and transforming growth factor-beta levels during experimental uveitis in the rabbit.

OBJECTIVE: To determine changes in the levels of nitric oxide metabolites and transforming growth factor-beta (TGF-beta) in the rabbit aqueous humour during ocular inflammation. DESIGN: Active experimental uveitis was induced by injection of porcine lens protein (PLP) in three rabbits and of human serum albumin (HSA) in three rabbits; three control rabbits received an injection of saline. OUTCOME MEASURES: Degree of inflammation, antibody titres (determined with the enzyme-linked immunosorbent assay), and aqueous humour levels of nitric oxide metabolites and TGF-beta. A modified Griess assay for nitrites and nitrates (NO2- and NO3-) was used as a measure of nitric oxide generation, and a modification of the CCL-64 mink lung epithelial cell bioassay was used to quantify TGF-beta levels. RESULTS: Following the primary immunologic challenge both experimental groups initially showed a two- to fourfold increment in aqueous levels of nitric oxide metabolites and TGF-beta compared with baseline values. At the peak of the clinically observed inflammation there was a significant increase in the mean nitric oxide metabolite level compared with the control value (p < or = 0.005) (432 nmol/mL for the PLP group and 112 nmol/mL for the HSA group) and a significant decrease (p < or = 0.03) in the mean TGF-beta level (3.1 ng/mL and 0.3 ng/mL respectively). CONCLUSIONS: Nitric oxide may be used as a marker for intraocular inflammation. The increased production of nitric oxide may reflect the loss of immunologic privilege of the ocular microenvironment that occurs during inflammation.

Analysis of gene expression during experimental uveitis in the rabbit.

OBJECTIVE: To analyse the pattern of gene expression at the messenger RNA level during ocular inflammation in the rabbit. DESIGN: A gene screen assay was used to quantify specific binding over background (expression index) of various activation markers and cytokines in rabbit iris-ciliary body obtained during active experimental uveitis induced by injection of porcine lens protein (two animals) and in a control group (two animals). OUTCOME MEASURES: Expression indices corresponding to the activation markers and cytokines assayed. RESULTS: Compared with the control eyes, analysis of triplicate samples from the inflamed eyes showed a significantly higher expression index corresponding to the proto-oncogenes c-fos, c-jun and Ha-ras, interleukin-2 and heat shock protein Hsp27 and a significantly lower index corresponding to transforming growth factor-beta (TGF-beta) (p < 0.05). CONCLUSIONS: Active experimental lens-induced uveitis is associated with a significant rise in the gene expression of cellular activation factors and a decrease in an immunoprotective factor (TGF-beta) in the iris and ciliary body of the rabbit.

DNA index and S phase fraction in uveal malignant melanomas.

AIMS: To predict 5 year survival in patients with uveal malignant melanomas DNA indices were studied. METHODS: Using 45 paraffin embedded uveal malignant melanomas, the DNA index and S phase fraction of each tumour were the predictor variables recorded. RESULTS: Using the Cox proportional hazards model, aneuploid tumours and tumours which had an S phase fraction greater than 4% were significant predictors of early death. In order to demonstrate a biological gradient between a larger DNA index and shorter survival time, linear regression and transformed linear regression models were used. However, no such gradient could be demonstrated. CONCLUSION: Although this study shows promise for the use of DNA studies in the prognosis of uveal malignant melanoma, the exact role of these techniques remains to be determined.

Quantification of transforming growth factor-ß in aqueous humor.

The use of biological assays in research often creates complex Problems of data analysis. Due to the nature of bioassays which respond to the intricate interplay between multiple factors that may either increase or decrease the readout, simple arithmetic or standard linear regression analysis often fails to provide accurate values when extrapolation from a standard curve is employed. Non-linear polynomial analysis could provide a better way for expressing such complex relationships. Using a bioassay for transforming growth factor-beta (TGF-ß) employing the mink lung epithelium cell line CCL-64, the authors present a new way to analyze experimental data based on non-linear polynomial statistics to provide accurate quantitative results when other simpler techniques have failed. This technique for data analysis provided accurate consistent results with acceptably small experimental variance. Using this approach, the bioactive concentration of TGF-ß in normal rabbit aqueous humor was found to be 2.72 ± 0.61 ng/ml, that is, corresponding to 2. 72 ng of PURE TGF-ß2, when referring to a standard curve.

Transforming growth factor-ß levels in aqueous humor during experimentally induced uveitis.

The anterior chamber of the eye is known to be a site of immune privilege. Particularly, the aqueous humor (AqH) appears to possess unique immunoregulatory properties. The authors have previously shown that human AqH (HAqH) may increase or decrease the proliferation of different cell types. Although no single factor has been established as solely responsible for these effects, much attention has been given to the 24-30 kD fraction of AqH, where transforming growth factor-beta (TGF-ß) is found. The purpose of this study was to determine the changes occurring in the rabbit AqH (RAqH) in relation to intraocular inflammation. Heterologous lens or human serum albumin (HSA) immunization-induced uveitis models were used in two groups of New Zealand albino rabbits to study the relationship between uveitis and TGF-ß. AqH and serum samples were obtained serially before, during and after the induction of ocular inflammation. Systemic humoral immunity to HSA or lens antigens was monitored using enzyme-linked immunosorbent assay (ELISA). A mink lung epithelial cell (CCL-64) bioassay for TGF-ß was used to quantify the amount of this cytokine in RAqH. TGF-ß levels in RAqH increased fourfold after the first immunization. A sharp decrease in RAqH TGF-ß levels was found in association with the development of acute intraocular inflammation. The implications of this finding to the etiology of uveitis are discussed.

Short term effects of glucocorticoids upon hippocampal ultrastructure.

This study evaluated hippocampal cytoarchitecture in male Sprague-Dawley (225-250 g) rats treated with corticosterone (CORT) and in unhandled, unstressed rats. CORT-treated animals received 20 mg/kg CORT in sesame oil/day x 3 days; each dose produces levels in the upper physiologic range, similar to those produced by major stressors. Using quantitative electron microscopy, sections from regions Ca3b, Ca1, and dentate gyrus were examined. Surface density and surface area of Golgi and the number of Golgi-stacks increased following CORT-treatment (p less than 0.05, ANOVA). Paralleling these changes surface area of rough endoplasmic reticulum and number of cisternae also increased in neuronal perikarya (p less than 0.001, p less than 0.003 respectively). In contrast, surface densities of mitochondria, multivesicular bodies, and lysosomes were unaffected by CORT treatment. These data suggest that both pyramidal and granule cells of hippocampus are subject to short term effects of high physiological levels of CORT, and that these effects may involve stimulation of protein biosynthesis.

Internalization of (125I) alpha-bungarotoxin into rat suprachiasmatic nucleus neurons and dendrites.

The distribution and rate of internalization of (125I) alpha-bungarotoxin (alpha-BuTX) was evaluated by quantitative electron microscopic autoradiography in rat suprachiasmatic nucleus. The ultrastructural distribution of silver grains was studied by both line source and 50% probability circle analyses. Line source analysis demonstrated that with increasing time following intracerebroventricular infusion, ligand is internalized from neuronal membranes. At all time points, real grain distribution was significantly different from randomly generated hypothetical grains. Quantification of silver grain localization by probability circle analysis indicated that membrane bound sources were mainly associated with axo-dendritic appositions, regardless of the length of time the tissue was exposed to radioligand. The compartment containing synaptic terminals was the most enriched when comparing real to hypothetical grains. By eight hours after intracerebroventricular infusion of specifically labelled alpha-BuTX binding sites were most likely to be within neurons and dendrites. These studies demonstrate that the majority of alpha-BuTX binding sites remain membrane bound with respect to time and may be associated with synaptic transmission; a significant proportion of silver grains are internalized within SCN neurons and dendrites.