Luen Hwu

Characterization of a new ribotoxin gene (c-sar) from Aspergillus clavatus

A new ribotoxin, c-sarcin, was isolated from a culture of Aspergillus clavatus. A full-length genomic DNA (c-sar) coding for c-sarcin was cloned and sequenced. The deduced amino acid sequence showed a high homology to restrictocin and alpha-sarcin. The native toxin as well as the recombinant protein hydrolysed ribosomes and naked RNA. The genomic structure of the c-sar gene had an intron located between the coding sequences for secretory signal peptide and the mature protein. The intron contained a stretch of 38 adenines. The intron sequence of c-sar was different from that of restrictocin but resembled that of alpha-sarcin. There was 34% identity between the intron of c-sarcin and alpha-sarcin, and this similarity was further increased to 83% if the stretch of polyadenine was omitted.

Production of type II ribotoxins by Aspergillus species and related fungi in Taiwan

A molecular investigation was conducted on the production of type II ribotoxin of the species Aspergillus and related fungi in Taiwan. Species that carried ribotoxin were confirmed by (1) cross-reactivity to anti-alpha-sarcin serum; (2) Southern dot hybridization; (3) PCR amplification of genomic DNA with specific primers; and (4) analysis of ribotoxic activity. Five new strains, A. clavatus, A. oryzae var. effusus, A. ostianus, A. tamarii, and Neosartorya fischeri var. spinosa, were identified to contain an alpha-sarcin-like ribotoxin. These positive strains exhibit ribotoxic activity by cleaving ribosomes and generating an alpha-fragment.

Evaluation of lentiviral-mediated expression of sodium iodide symporter in anaplastic thyroid cancer and the efficacy of in vivo imaging and therapy.

Anaplastic thyroid carcinoma (ATC) is one of the most deadly cancers. With intensive multimodalities of treatment, the survival remains low. ATC is not sensitive to 131I therapy due to loss of sodium iodide symporter (NIS) gene expression. We have previously generated a stable human NIS-expressing ATC cell line, ARO, and the ability of iodide accumulation was restored. To make NIS-mediated gene therapy more applicable, this study aimed to establish a lentiviral system for transferring hNIS gene to cells and to evaluate the efficacy of in vitro and in vivo radioiodide accumulation for imaging and therapy. Lentivirus containing hNIS cDNA were produced to transduce ARO cells which do not concentrate iodide. Gene expression, cell function, radioiodide imaging and treatment were evaluated in vitro and in vivo. Results showed that the transduced cells were restored to express hNIS and accumulated higher amount of radioiodide than parental cells. Therapeutic dose of 131I effectively inhibited the tumor growth derived from transduced cells as compared to saline-treated mice. Our results suggest that the lentiviral system efficiently transferred and expressed hNIS gene in ATC cells. The transduced cells showed a promising result of tumor imaging and therapy.

P21-Driven Multifusion Gene System for Evaluating the Efficacy of Histone Deacetylase Inhibitors by In Vivo Molecular Imaging and for Transcription Targeting Therapy of Cancer Mediated by Histone Deacetylase Inhibitor

Overexpressed histone deacetylase (HDAC) activity has been linked with tumor initiation and progression that prompt the development of histone deacetylase inhibitors (HDACIs) as anticancer agents. HDACI was reported to be able to activate p21 promoter through the SP1 binding sites in the proximal region of p21WAF1/CIP1 promoter. In this study, we established a p21WAF1/CIP1 promoter–driven triple-fused reporter gene system (p21-3H) to evaluate the efficacy of HDACI and the ganciclovir (GCV)-mediated anticancer effect contributed by HDACI-induced and p21-driven truncated herpes simplex virus-1 thymidine kinase sr39 mutant (ttksr39) in vitro and in vivo. Methods: The p21-3H construct was generated and stably or transiently transfected into H1299 cell lines. These cells were treated with trichostatin A or vorinostat (suberoylanilide hydroxamic acid [SAHA]) to evaluate the activation of p21 promoter–driven reporter gene expression by in vitro confocal fluorescence microscopy, luciferase assay, 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (3H-FEAU) cellular uptake, in vivo bioluminescence imaging, and 9-(4-18F-fluoro-3-hydroxymethylbutyl) guanine (18F-FHBG) small-animal PET imaging. The therapeutic efficacy on p21-3H–expressing tumor xenografts was assessed by daily administration with SAHA (100 mg/kg intraperitoneally) or GCV (20 mg/kg) for 9 d, followed by tumor volume measurement. Results: On treatment with trichostatin A or SAHA, H1299 cells carrying p21-3H showed a significant increase of luciferase activity, cellular uptake of 3H-FEAU (Moravek), and DsRed expression. In vivo tumor xenografts carrying p21-3H also showed increased luciferase activity by luminescent imaging and enhanced accumulation of 18F-FHBG by small-animal PET imaging. Furthermore, when cells transfected with p21-3H or p21/PstI-3H (which lacks p53-binding sites) were treated, the increase of luciferase activity was similar in both groups, indicating that HDACI-induced p21 promoter activation is independent of p53. Both in vitro and in vivo results showed improved therapeutic effect by combined treatment of GCV and HDACI. Conclusion: We have established an HDACI-inducible, p21-driven reporter system that has the potential for evaluating the anticancer effect of HDACIs on cancer cells by multiple molecular imaging modalities. Furthermore, ttksr39 in a p21-3H reporter construct provides a potential combination with thymidine kinase–mediated gene therapy to optimize the therapeutic benefit of HDACI.

Quantitative Measurement of the Thyroid Uptake Function of Mouse by Cerenkov Luminescence Imaging

Cerenkov luminescence imaging (CLI) has been an evolutional and alternative approach of nuclear imaging in basic research. This study aimed to measure the 131I thyroid uptake of mouse using CLI for assessment of thyroid function. Quantification of 131I thyroid uptake of mice in euthyroid, hypothyroid and hyperthyroid status was performed by CLI and γ-scintigraphy at 24 hours after injection of 131I. The 131I thyroid uptake was calculated using the equation: (thyroid counts − background counts)/(counts of injected dose of 131I) × 100%. Serum T4 concentration was determined to evaluate the thyroid function. The radioactivity of 131I was linearly correlated with the CL signals in both in vitro and in vivo measurements. CLI showed a significant decrease and increase of 131I thyroid uptake in the mice in hypo- and hyperfunctioning status, respectively, and highly correlated with that measured by γ-scintigraphy. However, the percent thyroid uptake measured by CLI were one-fifth of those measured by γ-scintigraphy due to insufficient tissue penetration of CL. These results indicate that CLI, in addition to nuclear imaging, is able to image and evaluate the 131I thyroid uptake function in mice in preclinical and research settings.

The Action Mode of the Ribosome-Inactivating Protein α-Sarcin

Based on the tertiary structure of the ribosome-inactivating protein α-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected. In this study, we found that the head-to-tail interaction between the first amino β-strand and the last carboxyl β-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated β-sheet is indispensable for catalyzing both substrates, suggesting that α-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino β-hairpin and that of the loop L3 in α-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the β-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the β-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes.

Rational design of a triple reporter gene for multimodality molecular imaging.

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.