Low molecular weight cyclin E is associated with p27-resistant, high-grade, high-stage and invasive bladder cancer.

Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer.

Tumor biology: use of tiled images in conjunction with measurements of cellular proliferation and death in response to drug treatments.

Tumor growth is dependent on the balance between cell proliferation and cell death, and these events occur heterogenously within an individual tumor. We present a methodology that provides integrative information about cell kinetics, cell death, and cell growth within individual tumors in animals treated with cytotoxic chemotherapeutic agents. Using HCT-116 and NCI-H460 cells, human colonic adenocarcinoma and non-small cell lung cells, respectively, traditional xenograft studies were performed. The tumor-bearing animals were treated with cyclophosphamide (Cytoxan), gemcitabine (Gemzar), or mitomycin C, and extensive analysis of the tumors was studied. Cell kinetics were evaluated by measuring the apoptotic and proliferation indices. The ability to image an entire tumor section using "tiling" by creating a large montage from many high-resolution images makes it possible to identify regional differences within areas of tumor and to demonstrate differences in these tumor regions after treatment with selected chemotherapeutic agents. Two specific areas within tumors have been identified: (a) areas of viable cells within the cell cycle, determined by bromodeoxyuridine and/or morphological characteristics determined by hematoxylin staining; and (b) areas of necrosis determined by the absence of bromodeoxyuridine and proliferating cell nuclear antigen-labeled cells coupled with morphological changes. By standardizing the tumor size to 100 mm2, different patterns of tumor responses to chemotherapeutic agents were determined. By creating such tiled images and by quantitating cell cycle kinetics, it is possible to gain a more complete understanding of tumor growth and response to treatment, leading to the development of more reliable methods for assessing the clinical behavior of anticancer drugs.

Inhibition of MAP kinase kinase (MEK) results in an anti-inflammatory response in vivo.

The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.

Novel small molecule alpha v integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors.

Recent evidence supports the involvement of integrins in angiogenesis: blockade of alpha v beta 3 and alpha v beta 5 integrins disrupts angiogenesis leading to decreased blood vessel formation and hence decreased tumor growth. We hypothesized that av antagonists could inhibit tumor growth in tumor cells devoid of alpha v beta 3 integrins. We evaluated SM256 and SD983, novel small molecules that are specific av antagonists in mouse models of angiogenesis and tumorigenesis, and compared them with standards: TNP470, a fumagillin analog in the clinic, and flavopiridol, a cell cycle kinase inhibitor. In vitro SM256 was a selective alpha v beta 3 inhibitor with an IC50 = 4nM, and the affinity of SD983 against the mouse endothelial alpha v beta 3 integrin yielded an IC50 = 2nM and an IC50 = 54nM against alpha v beta 5. In the mouse Matrigel model of angiogenesis SM256 decreased blood vessel formation (hemoglobin content) with an ED50 = 0.055 ug/kg/day, tenfold more potent than TNP470. SG545, an ester of SD983, decreased blood vessel formation with an ED50 = 6 ug/kg/day, while flavopiridol ED50 = 18 ug/kg/day. In the mouse xenograft model, using human colon carcinoma RKO cells that do not express alpha v beta 3 but express alpha v beta 5, tumor growth was inhibited by SG545 (10 mg/kg/day) and flavopiridol (5 mg/kg/every other day) 40% and 70%, respectively (p < 0.05). Although the proliferative index (measured by BrdU incorporation) was not significantly changed with SM256, SG545 or flavopiridol (29-32%), the apoptotic index increased significantly (p < 0.05) in the SM256 and SG545-treated groups (2.3-2.7%) compared with controls (1.1%), suggesting increased cell death contributed to decreased tumor volumes. Neovascularization decreased with SM256 and SG545 treatment. The data demonstrate that potent selective av antagonist can target endothelial cells, tumor cells, inhibit angiogenesis and inhibit tumor growth.

Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II.

Prostaglandins have wide-ranging effects in the body and are thought to be important mediators of inflammation. Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis, and occurs in both constitutive (COX-1) and inducible (COX-2) isoforms. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is both inducible and the major isoform of inflammatory cells. Reduction of prostaglandin production by inhibition of cyclooxygenases appears to be the main mechanism of action of most non-steroidal anti-inflammatory drugs (NSAIDS). Here we present an animal model of COX-2 deficiency that was generated by gene targeting. Defects in null mice correlating with reduced viability included renal alterations, characteristic of renal dysplasia (100% penetrance), and cardiac fibrosis (50% penetrance). Female Cox-2-/- mice were infertile. COX-2 deficiency failed to alter inflammatory responses in several standard models, but striking mitigation of endotoxin-induced hepatocellular cytotoxicity was observed.

Efficacy of DMP 840: a novel bis-naphthalimide cytotoxic agent with human solid tumor xenograft selectivity.

DMP 840, a novel bis-naphthalimide, was evaluated for antitumor efficacy in several tumor models in mice. As measured by a tumor growth inhibition assay, i.v. administration of DMP 840 to athymic nude mice at doses at or below the maximum tolerated dose resulted in curative activity against four human solid tumor xenografts, MX-1 mammary carcinoma, CX-1 and DLD-2 colon adenocarcinomas, and LX-1 lung carcinoma, producing full or incomplete regressions and/or percent tumor growth inhibition of > or = 96%. The efficacy of DMP 840 in the models was dose dependent. The activity of DMP 840 against the human tumors surpassed that demonstrated by several clinically used and investigational anticancer agents. In long-term growth delay studies, DMP 840 induced full regressions in 20 of 20 mice bearing MX-1 tumors, and tumors in one-half of these mice remained regressed for over 5 months. In addition, DMP 840 was curative against exponentially growing DLD-2 tumors staged at 500 mg and MX-1 tumors staged at 1000 mg. The bis-naphthalimide was equally efficacious when administered i.v. or i.p. but was slightly less active after oral dosing. Against both the MX-1 mammary carcinoma and the DLD-2 colon adenocarcinoma, some measure of schedule dependence was observed; the optimum schedule was daily for 9 days. Against L1210 and P388 murine leukemias, DMP 840 demonstrated little or no activity and was inactive against B16 murine melanoma. Overall, these results suggest that DMP 840 may be a human solid tumor selective cytotoxic agent.

XB596, a promising bis-naphthalimide anti-cancer agent.

We have synthesized a promising class of bis-naphthalimide anti-tumor agents. A representative compound in this series, XB596, exhibits potent in vitro growth inhibitory activity against several human and murine leukemic and solid tumor lines in culture, with IC50 values ranging from 7.2 to 147.5 nM. XB596 was almost as equally growth inhibitory against three doxorubicin-resistant cell lines compared with their parental lines. Using a human tumor colony-forming assay, XB596 demonstrated cytocidal activity against fresh human tumors taken directly from patients, with 23 of 25 evaluable tumors responding to a continuous exposure of 1 microgram/ml of XB596. When L1210 cells were incubated with XB596 for 1 h, the incorporation of uridine and thymidine into RNA and DNA, respectively, was inhibited with IC50 values of 0.14 microM. DNA single-strand breaks, but not double-strand breaks, were detected in XB596-treated L1210 cells. XB596 bound to DNA with guanine-cytosine sequence selectivity as shown by an indirect ethidium bromide displacement assay. XB596 was shown to interact with DNA by a spectrophotometric titration assay, with an estimated binding constant of 4.7 +/- 2.2 +/- 10(6) M-1. XB596 unwound supercoiled DNA as measured by agarose gel electrophoresis. These data are consistent with XB596 being a DNA intercalator. In vivo, XB596 demonstrated good anti-tumor activity against two human solid tumors (DLD-2 colon adenocarcinoma and MX-1 mammary carcinoma) xenografted in nude mice, but has not demonstrated anti-leukemic activity. In summary, XB596 is a pre-clinical anti-cancer agent which interacts with DNA and demonstrates good in vivo anti-tumor activity against human solid tumor xenografts.

Effects of modulation of basic fibroblast growth factor on tumor growth in vivo.

BACKGROUND: Recombinant human basic fibroblast growth factor (rHu-bFGF) is known to stimulate proliferation in some tumor cells and to modulate tumor vascularization. PURPOSE: The purpose of this study was to examine the possible role of this agent in the development of tumors. The study was designed to determine the effects of modulating bFGF activity in vivo in tumor models from cell lines with different responses to bFGF and with different content and receptor levels of bFGF. METHODS: Two tumor cell lines (human DLD-2 colon carcinoma and rat C6 glioma) were characterized for bFGF content and bFGF receptor levels by Western blot analysis in cultured cells and by studies of [125I]rHu-bFGF binding to sections from xenografts grown in nude mice. Tumor cell proliferation was monitored after treatment with rHu-bFGF or the DG2 or DE6 IgG monoclonal antibody to rHu-bFGF in culture and in vivo. RESULTS: C6 cells exhibited 7800 high-affinity receptors for rHu-bFGF per cell (dissociation constant [Kd] = 46 pM), while DLD-2 cells lacked high-affinity receptors. rHu-bFGF stimulated [3H]thymidine uptake by C6 cells, but the addition of DG2 IgG prevented this stimulation; rHu-bFGF had no effect on [3H]thymidine incorporation by DLD-2 cells. C6 cells had higher levels of immunoreactive bFGF than did DLD-2 cells. The xenografts from both cell lines exhibited high-affinity [125I]rHu-bFGF binding that was concentrated on vascular-like structures. rHu-bFGF at a dosage of 0.25 mg/kg given intraperitoneally daily for 18 days caused a twofold increase in DLD-2 tumor weight but had little effect on the growth of C6 xenografts. In contrast, daily intravenous injections of DG2 IgG given to mice had no effect on DLD-2 tumor growth but reduced growth of C6 tumors by approximately 30%--a statistically significant difference. CONCLUSIONS: The addition of exogenous rHu-bFGF or of a neutralizing antibody resulted in significant alterations in tumor growth in vivo, which were specific for tumor type and bFGF characteristics. While some of these effects may be mediated by the bFGF-responsive endothelial cells of the tumor vasculature (DLD-2 colon carcinoma), others may result from inhibition of bFGF-dependent tumor cell proliferation (C6 glioma). IMPLICATIONS: Studies that measure tumor blood flow are necessary to confirm that these effects are mediated by changes in tumor vasculature.

Treatment of osteoradionecrosis combined with pathologic fracture and osteomyelitis of the mandible with electromagnetic stimulation.

Osteoradionecrosis, complicated by pathologic fracture and osteomyelitis, is difficult to treat. Electromagnetic stimulation therapy may present a solution to this problem. A case of a 66-year old woman, suffering from osteoradionecrosis, pathologic fracture and osteomyelitis of the mandible, 6 years after irradiation therapy and partial resection of the mandible for squamous cell carcinoma of the gingiva, treated by electromagnetic stimulation, is presented. After 9 months of treatment, the patient was asymptomatic. Radiographic examination and bone scintigraphy carried out upon termination of the treatment, proved that healing of osteoradionecrosis and osteomyelitis had occurred. As far as we know, no previous reports have been published regarding this type of treatment.

RIA examinations of CSF hormones as a method of demonstrating leakage through the blood-brain and brain-CSF barriers.

Radioimmunoassay determinations of the levels of total T3, total T4, TSH, and prolactin in the CSF were performed on samples taken from 36 healthy individuals. The obtained reference values are the first of their kind. It is considered that RIA determinations of CSF hormone levels may provide a sensitive method for demonstrating pathological leakage through the blood-brain and brain-CSF barriers.

Direct subdural scintigraphy. Report of a case with subdural effusion following pneumococcal meningitis.

We report a case of pneumococcal meningitis in an 8 weeks olf female infant followed by persistent subdural effusion successfully treated by repeated subdural taps. The initial delineation of the subdural effusion, the decrease in size and the disappearance of the fluid were demonstrated by direct subdural scintigraphy (D.S.S.). The literature of pneumococcal meningitis, its diagnosis, treatment and complications are reviewed, and it is suggested that direct subdural scintigraphy should be employed as a diagnostic aid in the evaluation and follow up of subdural effusions.

Blood-C.S.F. barriers dysfunction in the chronic organic brain syndrome; a R.I.A. study.

C.S.F. samples of 35 patients, who suffered from verified chronic, non-tumorous organic brain syndrome, were radioimmunoassayed for T4 and T.S.H., and were compared to C.S.F.-R.I.A. samples from a control group of patients who underwent myelography because of lumbar disc. In addition T4 and T.S.H. plasma levels were evaluated in the O.B.S. patients. C.S.F. T4 and T.S.H. levels were significantly higher in 65% of the O.B.S. group of patients than those of the control group. The average determinations for T4 were: 0.77 muh/100 ml in O.B.S. group as against 0--0.4 micrograms/100 ml in the C.S.F.'s of the control group. P greater than 0,001 T.S.H. C.S.F. levels were 1.33 microU/ml in the O.B.S. group, and 0--0.6 microU/ml in the control group (P greater than 0.005). It is suggested that the elevated R.I.A. values of these hormones in the C.S.F. of the O.B.S. patients reflect a disruption of blood-C.S.F. barriers. Therefore in the organic brain syndrome there seems to exist a pathophysiological dysfunction of brain barriers in addition of the neural damage.

Thoracoscintigraphy in the differential diagnosis of cardiomegaly.

"Thoracoscintigraphy" is a radioisotopic scanning technique for demonstrating the nature of an enlarged cardiac shadow found on chest X-ray. The technique consists of superimposing scintiscans of the cardiac blood pool, the liver and the lungs and comparing the outline of the cardiac blood pool image with the cardiac shadow seen on X-ray. With this procedure the correct diagnosis of pericardial effusion was made in 11 of 15 patients and of pure cardiac dilatation and/or myocardial hypertrophy in three patients. In one of the patients the thoracoscintigraphic diagnosis was equivocal. Thoracoscintigraphy clearly demarcates the cardiac shadow from the surrounding organs, delineates the intracardiac blood pool within the cardiac shadow and indicates the best approach for pericardiocentesis. The procedure is harmless and noninvasive, and the radiation exposure is low and safe.

Triiodothyronine radioimmunoassay and its application to thyroid disorders.

An accurate, highly sensitive triiodothyrone (T3) radioimmunoassay system is described. Direct measurement in serum is made possible by the use of T3-free serum for standards, and blocking of T3 binding to thyroxine (T4)-binding globulin with salicylate. T4 cross reactivity was less than 0.3%. Mean T3 levels in 55 euthyroid, 32 hyperthyroid and 19 primary hypothyroid patients were 1.46 +/- 0.17, 5.34 +/- 1.86 and 0.49 +/- 0.34 (SD) ng/ml, respectively. T3 was found to be a more sensitive index than T4 in hyperthyroidism; in hypothyroidism it overlapped with euthyroidism. The presence of goiter had no effect on T3 concentrations in euthyroid patients. Among the patients studied, eight were found to have T3 toxicosis, including two patients with recurrent thyrotoxicosis. A significant T3 elevation was also found in euthyroid patients after thyroidectomy whereas T4 remained normal. The extent of T4 and T3 elevation was similar during the initial phase of subacute thyroiditis. There were significantly lower T3 values and higher thyroid-stimulating hormone values in umbilical cord blood than in maternal blood. In two euthyroid subjects, thyrotropin-releasing hormone stimulation caused a 50% rise in T3 levels and no change in T4 levels over a 2-h period.

Head and brain scan findings in rhinocerebral mucormycosis: case report.

Brain and bone scan findings in two patients suffering from rhinocerebral mucormycosis following kidney transplantation are presented. Two patients who had had kidney transplants and were sustained for over a month on immunosuppressive drugs developed a rare type of opportunistic infection--mucormycosis. They were examined in various stages of their disease. Special attention was paid to the scintillagraphic findings.