Detection of MDM2 alterations in cultured human hepatocytes treated with 17beta-estradiol or 17alpha-ethinylestradiol

BACKGROUND: Preneoplastic and neoplastic lesions of the liver are suspected to arise as a result of estrogen treatment. Here we present the first report on the modulational effects of the steroids 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) on oncogene MDM2 in human hepatocytes. MATERIALS AND METHODS: Collagen-embedded cultures of hepatocytes stimulated with different E2/EE2 concentrations were analyzed by immunocytochemistry, RT-PCR and sequencing for MDM2 protein/mRNA expression, MDM2 mRNA splicing and MDM2 gene mutation. RESULTS: The hepatocytes responded to stimulation with steroid E2/EE2 concentrations from 1-100 nmol/l with the overexpression of MDM2 protein while non-stimulated cells were negative. Stimulation with 1 nmol/l E2 and 10-100 nmol/l EE2 induced MDM2 splicing variants. Hepatocytes treated with 100 nmol/l E2 contained full-length MDM2 mRNA carrying a new type of MDM2 gene mutation. Unstimulated hepatocytes revealed neither mRNA splicing nor alteration of the MDM2 genes. CONCLUSION: The data show that steroid hormones are involved in the induction of MDM2 alterations in benign human hepatocytes. We speculate that some of the alterations may influence MDM2 function, thus possibly favouring genesis of liver changes.     

Inhibition of angiogenesis in vitro by alphav integrin-directed antisense oligonucleotides

The integrin alpha v beta 3 plays a central role in angiogenesis. In this study, we used antisense oligodeoxyribonucleotides (ONs) directed against the alpha v subunit of alpha v beta 3 to inhibit integrin expression. Ten ON sequences, which were selected by systematic alignment of computer-predicted secondary structures of alpha v mRNA, were transfected into human umbilical vein endothelial cells (HUVECs). Following stimulation by PMA, five antisense ONs significantly inhibited alpha v mRNA and protein expression in activated HUVEC at a concentration of 0.05 mciroM with complete prevention of PMA-induced alpha v up-regulation by the most potent antisense ON. Inhibition of alpha v expression was associated with significant inhibition of migration of HUVEC by 28% and had no effect on proliferation and apoptosis. Moreover, transfection of antisense ON inhibited the formation of tube-like structures of HUVEC in Matrigel by 44%. In a cell culture model of angiogenesis consisting of a co-culture of endothelial cells with fibroblasts, transfection of antisense ONs resulted in an inhibition of tube formation of 61%. In conclusion, alpha v antisense ONs are potent inhibitors of angiogenesis in vitro. They might, therefore, be a therapeutic alternative to antagonists, which directly bind to alpha v integrins, and might be useful for the treatment of malignant tumors and hematological malignancies.     

Technetium-99m-tetrofosmin scintigraphy in patients with metastatic soft tissue sarcoma

The lipophilic cationic compound Tc-99m-tetrofosmin has been demonstrated to be a valuable tool for the detection of a variety of tumours. Tc-99m-tetrofosmin uptake by sarcomas in vitro as well as in primary tumours has been reported. Data on the visualisation of metastatic soft tissue sarcomas using this tracer are missing so far. Ten consecutive patients with histopathologically verified metastatic soft tissue sarcoma were included in the present study. Five patients had previously received cytotoxic treatment, the other five patients were chemonaive. All patients underwent whole body planar examination after administration of 500-550 MBq Tc-99m-tetrofosmin, and in case of lung metastases on CT scan, SPECT images were carried out. Non-physiological accumulation of the tracer was considered as a positive result. Scintigraphic results were compared to conventional imaging by means of MRI/CT scanning. Visualisation of distant metastases was achieved in five patients all of whom were chemonaive, while in the chemotherapeutically pretreated patient group (n=5) false negative results were seen. Progressive disease was confirmed by follow-up in all patients. Pulmonary metastases were visualised only in SPECT acquisition and not on planar images. In one patient with diffuse bone marrow infiltration (inflammatory myofibroblastic sarcoma) Tc-99m-tetrofosmin scintigraphy was positive, while CT showed a negative result. According to our results, detection of metastatic soft tissue sarcomas by Tc-99m-tetrofosmin scintigraphy was strongly dependent on the history of previous treatment of the patient. A positive finding before initiation of chemotherapy was not indicative for subsequent therapeutic response. In the staging of chemonaive patients with metastatic soft tissue sarcoma Tc-99m-tetrofosmin may provide some additional information.     

Activity of E2F-dependent promoters in bladder carcinoma cells and their use for tumour-specific targeting of p53-induced apoptosis

Inactivation of P53 and RB functions are crucial changes in bladder cancer (TCC). High-level re-expression of P53 elicits apoptosis in TCC cell lines, but also--as shown here--in normal uroepithelial cells. Compromised RB function is thought to cause increased activity of E2F-dependent promoters in carcinoma cells. Indeed, several, but not all E2F-dependent promoters were stronger in TCC lines than in normal cells, with the highest activities in cell lines lacking RB rather than p16INK4A. Re-expression of p53 from an E2F-dependent promoter suppressed clone formation and induced apoptosis in TCC lines as efficiently as expression from the stronger RSV-LTR or LINE-1 promoters. In normal cells, p53 expression from an E2F-dependent promoter was tolerated, whereas expression from both stronger promoters was lethal. Thus, specific E2F-dependent promoters allow adjustment of p53 expression to selectively induce apoptosis in TCC vs. normal uroepithelial cells. This approach could be useful in targeting apoptosis to TCC and other carcinomas lacking p53 and RB function.     

Treatment of advanced gastric cancer with etoposide,folinic acid,and fluorouracil in the clinical setting: efficacy of therapy and value of serum tumor markers

The combination of etoposide, folinic acid, and 5-fluorouracil (5-FU) (ELF regimen) has been proved to be an active chemotherapy in patients with advanced gastric cancer. The aim of this study was to confirm the efficacy in the clinical setting and to correlate response with different parameters like serum tumor markers. We treated 60 patients with advanced gastric cancer with 120 mg/m² etoposide, 300 mg/m² folinic acid, and 500 mg/m² 5-FU, on d 1–3. The cycle was repeated on d 21. Objective response was obtained in 23% of all patients with measurable disease. Stable disease was obtained in 37%. The tumor-growth-control rate in patients with proximal carcinoma was significantly higher than in those with distal carcinoma (85% vs 48%, p=0.04). Median survival for all patients was 8.0 mo (95% confidence interval [CI] 7.0–8.5). In responsive patients, survival was more than two-fold longer than in patients with progressive disease. The administration of ELF could be performed safely on an outpatient basis. Toxicity was rather mild. The most frequently elevated serum tumor marker was CA 72-4 (55% of the patients). An elevated level of carcinoembryonic antigen before treatment was significantly correlated with progressive disease. A more than two-fold elevation of at least one marker under treatment was significantly correlated to progressive disease (p<0.002). A reduction of at least one marker under treatment was significantly correlated to tumor growth control (p<0.00015). The results of the present trial confirm the efficacy and low toxicity of the ELF regimen in advanced gastric carcinoma. Serum tumor markers proved suitable parameters for assessing response to treatment.     

Cytoplasmic localization of wild-type p53 in glioblastomas correlates with expression of vimentin and glial fibrillary acidic protein

Cytoplasmic accumulation of wild-type p53 in tumor cells indicates that the tumor suppressor is inactive with regard to growth suppressive functions. Whether this occurs randomly during tumor development or characterizes a certain tumor cell subset is not known. Here we assayed primary glioblastomas for expression and subcellular localization of p53 and determined a correlation with expression of intermediate filament proteins characterizing glial cell development. Sixty-nine percent of the tumors were p53 positive in immunohistochemistry. A significant number of tumors (23%) accumulated wild-type p53 in the cytoplasm, which correlated with the presence of vimentin and glial fibrillary acidic protein, except for 1 case. Tumors with exclusive nuclear p53 contained none or only one of these intermediate filament proteins. In an alternative approach, tumors positive for glial fibrillary acidic protein were screened for expression of p53 and vimentin. Thirty-eight percent of these tumors showed cytoplasmic p53, and all of those also expressed vimentin. Tumors with only nuclear p53 were vimentin negative, except for 1 case. No mutation was detected in p53 exons 5 to 8 in tumors with cytoplasmic p53, suggesting that they express wild-type p53. The data indicate that a cytoplasmic accumulation of wild-type p53 in human primary glioblastomas correlates with a certain intermediate filament protein expression, suggesting that it identifies a certain subset of tumors.     

Polysaccharides isolated from Echinacea purpurea herba cell cultures to counteract undesired effects of chemotherapy--a pilot study

In an open prospective study with matched historical controls we aimed to evaluate whether a polysaccharide fraction isolated from the herb Echinacea purpurea could counteract the undesired effects of chemotherapy. Fifteen patients with advanced gastric cancer undergoing palliative chemotherapy with etoposide, leucovorin and 5-fluorouracil (ELF) received for 10 days (beginning 3 days before chemotherapy) daily i.v. injections of 2 mg of a polysaccharide fraction isolated from Echinacea purpurea herb cell cultures (EPS-EPO VIIa). The median number of leukocytes 14-16 days after chemotherapy was 3630/microL (range 1470-5770) in the patients receiving EPS-EPO VIIa compared with 2370/microL (870-3950) in the patients of the historical control group (p = 0.015). EPS-EPO VIIa had no clinically relevant effects on phagocytic activity of granulocytes or on lymphocyte subpopulations. Sixty-eight adverse events including two deaths were observed, most likely due to chemotherapy and the general condition of the patients. However, an association with the test intervention cannot be ruled out completely. The results of this pilot study suggest that EPS-EPO VIIa might be effective in reducing chemotherapy-induced leukopenia. The efficacy and safety should be investigated in further studies.     

An overview of the pharmacology and pharmacokinetics of the newer generation aromatase inhibitors anastrozole,letrozole, and exemestane

BACKGROUND: The newer generation, nonsteroidal aromatase inhibitors (AIs) anastrozole and letrozole have shown superior efficacy compared with tamoxifen as first-line treatments and compared with megestrol acetate as second-line therapy in postmenopausal women with advanced breast carcinoma. In an open-label, Phase II trial, it was reported that exemestane showed numerical superiority compared with tamoxifen for objective response and clinical benefit. Because these agents ultimately may be administered for periods of up to 5 years in the adjuvant setting, it is of increasing importance to assess their tolerability and pharmacologic profiles. METHODS: In the absence of data from direct clinical comparisons, the published literature was reviewed for the clinical pharmacology, pharmacokinetic characteristics, and selectivity profiles of anastrozole, letrozole, and exemestane. RESULTS: At clinically administered doses, the plasma half-lives of anastrozole (1 mg once daily), letrozole (2.5 mg once daily), and exemestane (25 mg once daily) were 41-48 hours, 2-4 days, and 27 hours, respectively. The time to steady-state plasma levels was 7 days for both anastrozole and exemestane and 60 days for letrozole. Androgenic side effects have been reported only with exemestane. Anastrozole treatment had no impact on plasma lipid levels, whereas both letrozole and exemestane had an unfavorable effect on plasma lipid levels. In indirect comparisons, anastrozole showed the highest degree of selectivity compared with letrozole and exemestane in terms of a lack of effect on adrenosteroidogenesis. CONCLUSIONS: All three AIs demonstrated clinical efficacy over preexisting treatments. However, there were differences in terms of pharmacokinetics and effects on lipid levels and adrenosteroidogenesis. The long-term clinical significance of these differences remains to be elucidated.     

Expression of a "self-"antigen by human tumor cells enhances tumor antigen-specific CD4(+) T-cell function

CD4(+) T cells can recognize "self" tumor antigens, but the impact of tumor cell expression of self-antigens on CD4(+) T-cell function in humans is unknown. Here, we identify a new epitope (ISPNSVFSQWRVVCDSLEDYD) derived from tyrosinase-related protein-1 (TRP-1) using a predictive algorithm and mice transgenic for a chimeric HLA-DRB1*0401 molecule. We then compared the functions of TRP-1-epitope-specific, CD4(+) T-cell responses in normal healthy individuals to those found in patients with metastatic malignant melanoma. Surprisingly, we found that tumor-bearing patients had significantly higher levels of TRP-1-specific, CD4(+) T-cell function than healthy volunteers as measured ex vivo. Thus, the net effect of "self" antigen expression by tumor cells was the enhancement of tumor antigen-specific CD4(+) T-cell function, rather than immunosuppression. These findings indicate that antigens expressed by malignant melanoma cells can partially activate CD4(+) T lymphocytes.