The suppression of MUC1 expression in cultured cholangiocarcinoma cells using antisense MUC1 ribozyme strategy.

Ribozyme strategy has been employed to suppress the expression of target genes with a better efficacy than that of a regular antisense RNA strategy due to its favorable stoichiometry of the hybridization. A plasmid capable of controlling the expression of a portion of antisense RNA whose sequence is complementary to the 5' region of the coding sequence of MUC1 was constructed to include the requisite sequence of a hammerhead ribozyme. This plasmid is intended to suppress MUC1 expression. This novel plasmid was examined to see whether it functioned inside the harboring cell. Cultured cholangiocarcinoma cells prepared from the sediment of intrahepatic biliary fluid were employed as the transfection target since these cells have been confirmed to have high levels of MUC1 expression. The suppression of MUC1 expression in these cells after stimulation of the plasmid function for 16 h was demonstrated using flow cytometric analysis.

Expression of cyclooxygenase-1 and -2 in cultured human glioma cells of various stages.

Cyclooxygenase-1 (COX-1) and -2 (COX-2) are two isoforms of enzymes responsible for the biosynthesis of several forms of prostaglandins (PGs) from arachidonic acid. COX-1 is constitutively expressed in most normal tissues while COX-2 expression is regulated by certain stimuli such as cytokines and growth factors. The expression of COX-2 has been associated with many pathological conditions such as atherosclerosis, inflammation, and cancers (e.g. colon, breast, and lung cancers). COX-2 expression may be associated with the progression of cancers since PGE2, a major product of both isoforms was identified as a tumor-derived immune suppressor. Therefore, the suppression of COX-2 activity using specific COX-2 inhibitors (recently classified non-steroidal anti-inflammatory drugs) may delay the progression of tumors harboring COX-2. The search for tumors that highly express both isoforms of COX serves as a guide to target tumors that are most likely to be susceptible to treatment with specific COX-2 inhibitors. To study a homogeneous population of tumor cells, fifteen samples of primary culture cells were prepared from different human brain tissues and tumors collected from subjects operated on at Siriraj Hospital. Cells were grown to confluent in 75-cm2 tissue culture flask for about 4 weeks. After which time, cells were extracted to evaluate COX-1 and COX-2 protein expressions by immunoblot using specific antibodies. Four out of seven samples of glioblastoma multiforme cells strongly expressed COX-2, while all samples of examined cultured cells expressed COX-1. Cultured cells from astrocytomas had only faint staining for COX-2, while maintaining strong COX-1 expression. Thus COX-2 expression was limited to samples of glioblastoma multiforme, the most advanced stage of astrocytoma. Further study for the suppression of COX-2 activity in inducing tumor apoptosis and in improving cellular immunity against this advanced cancer should be elucidated.

Expression of insulin-like growth factor I and II in various regionally important solid tumors.

Insulin-like growth factor I (IGF-I) may thwart a T cell response to several cancers, since suppression of IGF-I expression using an episome transcribing an antisense RNA into rat glioma and murine teratocarcinoma, which are known to actively express IGF-I, resulted in tumor regression and CD8+ T cell infiltration.1,2,3,4 This finding led to the gene therapy protocol for human glioblastoma multiforme, a known IGF-I over-expressing tumor, using the same strategy in 1993. The hypothesis that cancers which over-express IGF-I can be cured by this episome has led us to search for other cancers which actively produce IGF-I. Many cancers were reported to express IGF-I4, but most of the geographically important tumors in Thailand had not been studied for the expression of IGF-I. Our immediate goal was to survey the leading human cancers in the Tumor Registry of Siriraj Hospital (i.e., non-small cell lung cancer, cholangiocarcinoma, hepatocellular carcinoma, papillary thyroid carcinoma, nasopharyngeal carcinoma) including all stages of astrocytomas for the expression of IGF-I and IGF-II (a homologue of IGF-I). Tumor tissue collected from paraffin blocks deposited at the Department of Pathology, Siriraj Hospital was screened for the presence of the predominant form of IGF's (IGF-I or -II). Tumor tissues that express any specific form of IGF's more than adjacent wild-type cells are potential candidates for an antisense gene therapy against the expression of that specific form of IGF. Paraffin sections containing these cancer cells and adjacent wild-type cells were examined by immunohistochemical technique using mouse IgG-1 antihuman IGF-I, or antihuman IGF-II as primary antibodies. Normal liver tissue, known as the largest producer of both forms of IGF's, was used as a positive control. The staining pattern of both IGF-I and IGF-II in the liver was highest in the cytoplasm of normal hepatocytes. The majority of the tumors except for papillary thyroid carcinoma and nasopharyngeal carcinoma strongly expressed IGF-I. Only a few samples of the tissues studied expressed a low level of IGF-II, if any. This raises a further working proposal that the blockade of IGF-I synthesis in these tumors may help to delay the tumor progression so that an effective immune response can be established and destroy the tumor.

Satisfaction in use examination results for computer-assisted instruction on antiarrhythmic drugs.

Computer-assisted instruction (CAI) has become a modern tool for self-instruction in subjects that were once thought to be erudite. We have used CAI in teaching several topics of pharmacology to third year medical students since 1995. In 1996, we developed CAI in antiarrhythmic drugs by using the software ToolBook, version 3.0, comprising 29 electronic pages. Most pages contain English text, drawn images, animation and sound effects. To minimize the size of CAI, no scanned images or video clips were used. Some preliminary explanations were given before the class was held to enable the students to use CAI as provided. Subjective satisfaction after using CAI was evaluated from questionnaire answered immediately after the examination. An objective evaluation was made from the number of correct answers to eight antiarrhythmic questions in the third sectional examination (which contained 85 questions). From the questionnaire, the students are divided into two groups. The first group comprised 83 questions who had never used CAI; the second group contained 123 students who had used it at least once. The mean number of correct answers+SD to the antiarrhythmic questions in the first and the second groups were 3.60+1.51 and 4.14+1.73, respectively, which shows a atatistically significant difference (P<0.01) despite the similarity of the original scores in the first two exams (70.27+8.85 percent and 70.57+9.81 percent, respectively. CAI therefore helps to increase the number of correct answers to antiarrhythmic questions, even though the increase is small. The difference may have resulted from including two students who answered all the questions wrong and another student who answered all the questions correctly in the second group as well as English being the only language used in CAI.