Cellular Response to Efficient dUTPase RNAi Silencing in Stable HeLa Cell Lines Perturbs Expression Levels of Genes Involved in Thymidylate Metabolism

dUTPase is involved in preserving DNA integrity in cells. We report an efficient dUTPase silencing by RNAi-based system in stable human cell line. Repression of dUTPase induced specific expression level increments for thymidylate kinase and thymidine kinase, and also an increased sensitization to 5-fluoro-2′-deoxyuridine and 5-fluoro-uracil. The catalytic mechanism of dUTPase was investigated for 5-fluoro-dUTP. The 5F-substitution on the uracil ring of the substrate did not change the kinetic mechanism of dUTP hydrolysis by dUTPase. Results indicate that RNAi silencing of dUTPase induces a complex cellular response wherein sensitivity towards fluoropyrimidines and gene expression levels of related enzymes are both modulated.     

Cellular response to efficient dUTPase RNAi silencing in stable HeLa cell lines perturbs expression levels of genes involved in thymidylate metabolism

dUTPase is involved in preserving DNA integrity in cells. We report an efficient dUTPase silencing by RNAi-based system in stable human cell line. Repression of dUTPase induced specific expression level increments for thymidylate kinase and thymidine kinase, and also an increased sensitization to 5-fluoro-2'-deoxyuridine and 5-fluoro-uracil. The catalytic mechanism of dUTPase was investigated for 5-fluoro-dUTP. The 5F-substitution on the uracil ring of the substrate did not change the kinetic mechanism of dUTP hydrolysis by dUTPase. Results indicate that RNAi silencing of dUTPase induces a complex cellular response wherein sensitivity towards fluoropyrimidines and gene expression levels of related enzymes are both modulated.     

PAF and Haematopoiesis. I. 5-Fluoro-Uracil Induces PAF Production in Haematopoietic Organs of Rats

Haematopoietic organs of rats were examined for the presence of platelet-activating factor (PAF) and acetylhydrolase before and after treatment with 5-fluoro-uracil (5-FU) (200 mg/kg) a chemotherapeutic compound with apoptotic effects. PAF was reported in thymus, spleen and femoral bone marrow of rats with or without 5-FU. Although acetylhydrolase activity in organs was not affected by 5-FU treatment, elevated levels of PAF were observed in thymus and spleen. For the first time PAF is reported in haematopoietic organs of rats, strengthening in vitro data suggesting its role in the apoptotic processes in thymus, in the modulation of the immune response, and in the regulation of haematopoiesis.     

Cardiac involvement in central lung cancer

An x-ray picture of central lung cancer was analyzed in 34 operated patients in whom tumors had infiltrated the myocardium (left atrial infiltration in 33, right atrial infiltration in one, ventricular infiltration in 2). The comparison of x-ray findings with gross specimens of the removed lungs showed that tumor mass consisted of a primary tumor and metastatic bronchopulmonary lymph nodes that made impossible the differentiation of separate elements of the root x-ray image. Angiography was performed in 15 patients with suspected infiltration of the heart by a tumor on the basis of x-ray findings. Cardiac affection which was characterized on x-ray by change in the shape of the chambers, irregularity of their outlines or a filling defect at the site of tumor infiltration. Suspected cardiac infiltration by a tumor can be based on indirect x-ray signs: the presence of a large tumor mass in the area of the root, a shift of the deflated lung regions to the heart, the absence of an image of heart borders and pulmonary veins on tomograms, and a confluent image of the affected lung and cardiac shadow. These signs serve an indication for angiography.     

A WKYMVm-containing combination elicits potent anti-tumor activity in heterotopic cancer animal model

The development of efficient anti-cancer therapy has been a topic of intense interest for several decades. Combined administration of certain molecules and immune cells has been shown to be an effective form of anti-cancer therapy. Here, we examined the effects of administering an immune stimulating peptide (WKYMVm), 5-fluoro-uracil (5-FU), and mature dendritic cells (mDCs) against heterotopic cancer animal model. Administration of the triple combination strongly reduced tumor volume in CT-26-inoculated heterotopic cancer animal model. The induced anti-tumor activity was well correlated with FAS expression, caspase-3 activation, and cancer cell apoptosis. The triple combination treatment caused recruitment of CD8 T lymphocytes and natural killer (NK) cells into the tumor. The production of two cytokines, IFN-γ and IL-12, were strongly stimulated by administration of the triple combination. Depletion of CD8 T lymphocytes or NK cells by administration of anti-CD8 or anti-asialoGM1 antibody inhibited the anti-tumor activity and cytokine production of the triple combination. The triple combination strongly inhibited metastasis of colon cancer cells in a heterotopic cancer animal model as well as in a metastatic cancer animal model, and enhanced the survival rate of the mice model. Adoptive transfer of CD8 T lymphocytes and NK cells further increased the survival rate. Taken together, we suggest that the use of triple combination therapy of WKYMVm, 5-FU, and mDCs may have implications in solid tumor and metastasis treatment.     

Roentgenologic characteristics of small bronchoalveolar cancer of the lung under conditions of image processing using linear filtration (roentgenomorphologic comparison)

X-ray and morphological signs were compared in 50 patients with histologically verified bronchioloalveolar cancer (BAC). Particular attention was drawn to a study of an x-ray picture after computer processing of a tomographic image using a special technique. The most characteristic x-ray signs of BAC were subpleural localization, the presence of a fragmented scar in a tumor, and ill defined outlines. The peculiarities of an x-ray picture of small-size BAC were determined by the morphological structure of a tumor, a degree of its differentiation that could be detected in most cases only after image processing by the linear filtration technique.     

X-ray diagnosis of the spread of cancer of the esophagus to the trachea and major bronchi

X-ray and endoscopic investigations were compared in 80 operated on patients to estimate the role of the x-ray method for a choice of adequate therapeutic tactics for esophageal cancer patients. Operations were explorative or palliative resulting from the involvement of the tracheobronchial tree in a tumor process. The efficacy of the x-ray method in the detection of tracheal compression by an esophageal tumor is higher than that of tracheobronchoscopy which in its turn, has its advantages in the determination of tumor spreading to the left major bronchus.     

19F NMR of RNA. The Structural and Chemical Aspects of 5-Fluoro-cytidine and-uridine Labelling of Oligoribonucleotides

Abstract

Results of PM3 semiempirical calculation revealed that energy and hydrogen bonds geometry of 1-methyl-5-fluoro-uracil and -cytosine base-pairs with 9-methyl-adenine and -guanine respectively are virtually the same as for the natural bases. Analysis of proton coupling constants proved that the sugar puckering of 5-fluorouridine and 5-fluorocytidine is analogous to non-modified ribonucleosides. 5-Fluorocytidine was regioselectively introduced to oligoribonucleotides, prepared using 2′-O-tert-dimethylsilyl protection, via post-synthetic quantitative ammonolysis of 4-O-methyl-5-fluorouridine derived precursor.

     

Magnetic resonance tomography of malignant tumors of the maxilla]

The authors discussed their experience in investigating 20 patients with maxillary malignant tumors using routine x-ray studies and MR-tomography, and 13 patients, investigated in the same way plus CT. MRT permitted defining a topical localization of a tumor and its spreading to adjacent anatomical regions (the pterygopalatine and infratemporal fossae, pharynx and peripharyngeal space, orbit, buccal soft tissues, and the cranial cavity). MRT was used to differentiate between tumor tissue and inflammation even within the sinus. As to the detection of osseous destruction, CT seemed much more superior than MRT. It was only in one patient that x-ray findings brought about better results than MRT because of artefacts resulting from metal crowns on the affected side.     

DNA polymerase and thymidine kinase activities in MC-29 virus-induced transplantable hepatoma and the effect of cytostatic treatment on these activities

The activity of DNA polymerases and thymidine kinase was compared in the MC-29 leukosis virus-induced transplantable hepatoma and in the livers of rats treated with cyclophosphamide (CP), cytosine-arabinoside (ara-C) and 5-fluoro-uracil (5-FU). The specific activity of DNA polymerase was twenty times greater in the MC-29 leukosis virus-induced hepatoma, while thymidine kinase was only 3–5 times greater than in the liver.All three enzymes showed Michaelis-Menten kinetics in their substrate and template saturation curves. The template utilization of DNA polymerases from hepatoma and from liver was compared. Both had higher activities on a poly(dA) · poly(dT) template at pH 8.0, than on DNA at pH 7.5. After chromatography on a phosphocellulose column two polymerases were separated. The first peak eluted by 0.15 M KCl preferred DNA as template (polymerase α). The second eluted by 0.5 M KCl worked better on poly(dA) · poly(dT) (polymerase β). Thymidine kinase was eluted by 0.25 M KCl. Inhibition by N-ethylmaleimide (NEM) showed the polymerase α to be sensitive and the polymerase β to be resistant to the sulfhydryl blocking agent; similar to the respective enzymes of other eukaryotic cells. The specific acitivity of DNA polymerase decreased after CP treatment at 6 h and 72 h and after ara-C treatment at 72 h. The specific activities of thymidine kinase were not changed significantly in response to the drug administrations.     

Measurement of tumor volumes improves RECIST-based response assessments in advanced lung cancer

OBJECTIVE: This study was designed to characterize the reproducibility of measurement for tumor volumes and their longest tumor diameters (LDs) and estimate the potential impact of using changes in tumor volumes instead of LDs as the basis for response assessments. METHODS: We studied patients with advanced lung cancer who have been observed longitudinally with x-ray computed tomography in a multinational trial. A total of 71 time points from 10 patients with 13 morphologically complex target lesions were analyzed. A total of 6461 volume measurements and their corresponding LDs were made by seven independent teams using their own work flows and image analysis tools. Interteam agreement and overall interrater concurrence were characterized. RESULTS: Interteam agreement between volume measurements was better than between LD measurements (ı = 0.945 vs 0.734, P = .005). The variability in determining the nadir was lower for volumes than for LDs (P = .005). Use of standard thresholds for the RECIST-based method and use of experimentally determined cutoffs for categorizing responses showed that volume measurements had a significantly greater sensitivity for detecting partial responses and disease progression. Earlier detection of progression would have led to earlier changes in patient management in most cases. CONCLUSIONS: Our findings indicate that measurement of changes in tumor volumes is adequately reproducible. Using tumor volumes as the basis for response assessments could have a positive impact on both patient management and clinical trials. More authoritative work to qualify or discard changes in volume as the basis for response assessments should proceed.     

Analysis of Potential Radiosensitizing Materials for X-Ray-Induced Photodynamic Therapy

For a development of deep tumor treatment in photodynamic therapy, a feasibility of novel radiosensitizers induced by x-ray was investigated. The sensitizers are designed to generate reactive oxygen species (ROS) inside or outside the cell, possibly leading to damage exclusively on tumor cells and reservation of normal cells along the x-ray path. Taking note of the similarity in energy transfer mechanism in photocatalysts, scintillators, and particulate semiconductors, we chose TiO₂, ZnS:Ag, CeF₃, and quantum dots (CdTe and CdSe) in particulate form, which contain heavy atoms for efficient absorption of x-rays. A parameter study for x-ray operating conditions showed that in a typical scenario, photons with 20 to 170 keV energy are attenuated by 90% through the region of particle dispersed aqueous solution at varying concentration between 0.01 and 100 wt%. The amount of ROS generation under the exposure of polychromatic x-ray was measured using dihydroethidium reagent which detects an integrated amount of several species. Proportional increase in ROS generation to x-ray dose was observed for varying concentrations of TiO₂, ZnS:Ag, CeF₃, and CdSe quantum dot dispersions. Then, HeLa cells were mixed with aqueous solutions dispersed with sensitizing materials at a concentration of 3.0 mg/ml and were exposed to x-ray. Their survival fraction obtained by a cell proliferation reagent WST-1 immediately after the irradiation showed insignificant effects of sensitizing materials except at large doses. To enhance the sensitization effect, bio-conjugated CdSe quantum dots were internalized in the cytoplasm up to a concentration of 1.0 ng/ml. The cells were irradiated by x-ray up to 5 Gy, and their survival fraction was measured by the colony forming ability 9 days after irradiation. Survival fraction of the cells treated with quantum dots were less than those without quantum dots for all doses, suggesting that the colony forming ability is impaired by the internalized quantum dots.     

Analysis of pre-treatment markers predictive of treatment benefit for the therapeutic cancer vaccine MVA-5T4 (TroVax)

Cancer vaccines such as MVA-5T4 (TroVax^?) must induce an efficacious immune response to deliver therapeutic benefit. The identification of biomarkers that impact on the clinical and/or immunological efficacy of cancer vaccines is required in order to select patients who are most likely to benefit from this treatment modality. Here, we sought to identify a predictor of treatment benefit for renal cancer patients treated with MVA-5T4. Statistical modeling was undertaken using data from a phase III trial in which patients requiring first-line treatment for metastatic renal cell carcinoma were randomized 1:1 to receive MVA-5T4 or placebo alongside sunitinib, IL-2 or IFN-??. Numerous pre-treatment factors associated with inflammatory anemia (e.g., CRP, hemoglobin, hematocrit, IL-6, ferritin, platelets) demonstrated a significant relationship with tumor burden and patient survival. From these prognostic factors, the pre-treatment mean corpuscular hemoglobin concentration (MCHC) was found to be the best predictor of treatment benefit (P?<?0.01) for MVA-5T4 treated patients and also correlated positively with tumor shrinkage (P?<?0.001). Furthermore, MCHC levels showed a significant positive association with 5T4 antibody response (P?=?0.01). The latter result was confirmed using an independent data set comprising phase II trials of MVA-5T4 in patients with colorectal, renal and prostate cancers. Retrospective analyses demonstrated that RCC patients who had very large tumor burdens and low MCHC levels received little or no benefit from treatment with MVA-5T4; however, patients with smaller tumor burdens and normal MCHC levels received substantial benefit from treatment with MVA-5T4.     

A Pilot Study of Diffusion-Weighted MRI in Patients Undergoing Neoadjuvant Chemoradiation for Pancreatic Cancer

PURPOSE: In the current study we examined the ability of diffusion MRI (dMRI) to predict pathologic response in pancreatic cancer patients receiving neoadjuvant chemoradiation. METHODS: We performed a prospective pilot study of dMRI in patients with resectable pancreatic cancer. Patients underwent dMRI prior to neoadjuvant chemoradiation. Surgical specimens were graded according to the percent tumor cell destruction. Apparent diffusion coefficient (ADC) maps were used to generate whole-tumor derived ADC histogram distributions and mean ADC values. The primary objective of the study was to correlate ADC parameters with pathologic and CT response. RESULTS: Ten of the 12 patients enrolled on the study completed chemoradiation and had surgery. Three were found to be unresectable at the time of surgery and no specimen was obtained. Out of the 7 patients who underwent pancreaticoduodenectomy, 3 had a grade III histopathologic response (> 90% tumor cell destruction), 2 had a grade IIB response (51% to 90% tumor cell destruction), 1 had a grade IIA response (11% to 50% tumor cell destruction), and 1 had a grade I response (> 90% viable tumor). Median survival for patients with a grade III response, grade I-II response, and unresectable disease were 25.6, 18.7, and 6.1 months, respectively. There was a significant correlation between pre-treatment mean tumor ADC values and the amount of tumor cell destruction after chemoradiation with a Pearson correlation coefficient of 0.94 (P = .001). Mean pre-treatment ADC was 161 × 10^− 5 mm²/s (n = 3) in responding patients (> 90% tumor cell destruction) compared to 125 × 10^− 5 mm²/s (n = 4) in non-responding patients (> 10% viable tumor). CT imaging showed no significant change in tumor size in responders or non-responders. CONCLUSIONS: dMRI may be useful to predict response to chemoradiation in pancreatic cancer. In our study, tumors with a low ADC mean value at baseline responded poorly to standard chemoradiation and would be candidates for intensified therapy.     

The SOS screen in Arabidopsis: A search for functions involved in DNA metabolism

The SOS screen, as originally described by Perkins et al. (1999) [7], was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS⁺ candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS⁺ candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS⁺ candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.     

X-ray-ultrasonic parallels in tumors of the stomach and intestines]

Combined clinical, radio and ultrasound investigation of 46 patients was carried out. It was shown that routine transabdominal UTS could be employed in diagnosis of gastric and intestinal tumors but mainly at more advanced stages (T3, T4). Potentialities of the method were shown to depend, to a great extent, on a tumor site, growth, type, sizes, and spreading, as well as on the resolving power of an US diagnostic apparatus. The use of a high resolution apparatus made it possible to detect tumor invasion, but topography of a tumor was difficult to define. The x-ray or endoscopic method can be used as an adjuvant method of specified diagnosis of gastric and intestinal tumors.     

131I]metaiodobenzylguanidine treatment of malignant pheochromocytoma: experience of the Rome group.

Five cases of malignant pheochromocytoma (3 men and 2 women, aged 26-43 years) were treated with [131I]metaiodobenzylguanidine (131I-MIBG). One patient had a voluminous adrenal tumor and multiple distant metastases; two patients had a recurrent tumor; two others a post-surgical residual tumor. The therapeutic procedure essentially consisted of single doses (2.6-7.4 GBq) of 131I-MIBG administered by slow i.v. infusion, given in several therapeutic courses at 1-5 month intervals. The treatment resulted in a complete response in one case with residual tumor and in a partial response in the case with disseminated disease. Two cases showed stabilization of the disease, whereas therapy was ineffective in the fifth case. Nevertheless, pain relief was observed in this patient. The treatment had a very low toxicity and was well tolerated by all patients.     

Synthesis, characterization, and antitumor activity of 5-iodouracil complexes.

Complexes of 5-iodouracil (5IU) with Mn(II), Co(II), Cu(II), Zn(II), and Cd(II) ions have been prepared, characterized, and subjected to a screening system for evaluation of antitumor activity against Sarcoma-180 (S-180) and L 929 tumor cells. The complexes were characterized by their elemental analysis, infrared spectra, electronic spectra, magnetic measurements, and powder x-ray diffraction. The antitumor activity results indicate that some complexes have good antitumor activity both in vivo and in vitro against S-180 and L 929 tumor cells.     

Treatment of a patient with B cell lymphoma by I-131 LYM-1 monoclonal antibodies

A patient with Richter's syndrome, a malignant lymphomatous transformation of chronic lymphocytic leukemia, had become moribund with rapidly enlarging masses, granulocytopenia and thrombocytopenia despite the use of conventional chemotherapy and radiotherapy. Greater than ten percent of a test dose of I-131 Lym-1, a murine monoclonal antibody produced against Burkitt's African B cell lymphoma, was accumulated by her tumor. The patient was subsequently treated with a series of injections of I-131 Lym-1 with dramatic clinical response, reduction of tumor volume by x-ray computerized tomography and progression of circulating cellular elements toward normality. Her course over the next ten months was not like that to be expected for Richter's syndrome, which has an average survival of four months. This mode of treatment appears promising.