Molecular tryst peeping: detection of interactions between nonlabeled nucleic acids by fluorescence resonance energy transfer.

We have developed a new method for monitoring the interactions between nonlabeled RNAs that involves detection of fluorescence resonance energy transfer (FRET) between two DNA probes with different fluorescent label. The sequences of the probes are complementary to those of the RNAs. In this study, we examined the interaction between a portion of the LTR RNA of HIV-1 and the corresponding antisense RNA. The antisense RNA was designed not to bind to the fluorescent DNA without prior hybridization to the target RNA. A mixture of RNAs and DNA probes with fluorescent labels was fractionated by electrophoresis on a nondenaturing polyacrylamide gel and then the gel was analyzed with a fluorescence imaging analyzer. FRET was observed only in the presence of target RNA, antisense RNA, and both of the fluorescent DNA probes. This strategy should be useful for the detection of interactions between nucleic acids that cannot be subjected to chemical modification, such as RNA transcripts inside cells.

Creatine kinase gene mutation in a patient with muscle creatine kinase deficiency.

BACKGROUND: We describe a 56-year-old woman admitted to the hospital with a diagnosis of acute myocardial infarction without an increase of serum creatine kinase (CK) activity during her clinical course. She died on the 11th hospital day, and the diagnosis was confirmed by autopsy. The patient had had no previous muscular symptoms. METHODS: Expression of the CK-muscle (CK-M) protein in cardiac tissue was examined by immunoblotting and immunochemical staining. CK-M mRNA expression was estimated by semiquantitative reverse transcription-PCR. Gene structure of CK-M was determined by Southern blotting and direct sequencing of 2251 bp. Existence of a point mutation in the CK-M gene was examined by restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) in the patient and in 108 controls. RESULTS: CK-M protein in the myocardial tissue of the patient was substantially lower (103 +/- 7 ng/mg protein) than in control myocardial tissue (35 800 +/- 2860 ng/mg protein). Immunoreactive CK-M in the patient tissue sample was 0.3% of the value for the control sample. CK-M mRNA was 53-fold less in the patient sample compared with the control. This very low expression of CK-M mRNA was considered to be the primary reason for CK-M deficiency. Direct sequencing revealed a point mutation at residue 54 in exon 2, which was specific for the patient. No other abnormalities were found in the CK-M gene of the patient. CONCLUSIONS: This report identifies a molecular abnormality in human CK deficiency and discusses the physiologic relevance of CK-M.

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

Expression of human telomerase catalytic subunit gene in cancerous and precancerous gastric conditions.

BACKGROUND AND AIMS: Telomerase activity is thought to be necessary for cellular immortality and carcinogenesis. The mRNA that encodes the telomerase catalytic subunit (hTERT) has recently been identified, and expression of hTERT mRNA is thought to regulate activation of telomerase. To determine at what stage of carcinogenesis cells begin to express hTERT, we analysed hTERT mRNA expression in gastric carcinoma and precancerous conditions, focusing on chronic gastritis with or without intestinal metaplasia. METHODS: Using reverse transcription-polymerase chain reaction, hTERT gene expression was investigated in 18 gastric cancers and 60 specimens of chronic gastritis. Telomerase activity was evaluated using telomeric repeat amplification protocol. RESULTS: Sixteen of 18 (89%) gastric carcinomas expressed hTERT mRNA, and this expression was unrelated to histological type or depth of invasion. Telomerase activity was found in seven of eight (88%) gastric cancer tissues, all of which expressed hTERT mRNA. Expression of hTERT mRNA was positive in 14 of 60 (23%) specimens of chronic gastritis, and was most prominent in seven of 15 (47%) specimens of gastric mucosa with intestinal metaplasia. Expression of the hTERT gene was significantly more frequent in chronic gastritis with intestinal metaplasia than in gastritis without intestinal metaplasia (P=0.030). In addition, hTERT gene expression was not correlated with age, sex, biopsy site, histological grade of inflammatory cells, glandular atrophy and lymph follicles, or infection with Helicobacter pylori. None of eight normal gastric mucosa expressed hTERT mRNA. CONCLUSIONS: Our results indicate that hTERT mRNA is expressed in precancerous conditions as well as in gastric cancer, and that hTERT gene expression is induced at an early stage of gastric carcinogenesis.

Control of the functional activity of an antisense RNA by a tetracycline-responsive derivative of the human U6 snRNA promoter.

In an effort to develop a regulatable derivative of the promoter of the human gene for U6 snRNA, we generated several constructs composed of the human U6 snRNA promoter and sequences derived from the gene for the tetracycline operator of a prokaryotic tetracycline resistance transposon. One of the constructs had strong transcriptional activity in the presence of tetracycline that was equivalent to 80% of the activity of the wild-type promoter. Furthermore, the transcriptional activity was almost completely repressed in the absence of tetracycline. Transcriptional activity became detectable within 4 hr after the addition of tetracycline to the culture medium. We used this system to control the functional activity of an antisense RNA for a chimeric gene derived from genes for the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP). A plasmid that expressed the chimeric gene and a plasmid that expressed the antisense RNA under the control of the inducible U6 promoter were used to cotransfect HeLa cells that were producing the tetracycline repressor protein (Tet R). Addition of tetracycline to the culture medium 12 hr after transfection resulted in the almost complete disappearance of the fluorescent signal due to the chimeric protein within 24 hr. Our results suggest that this expression system might be a useful tool for controlling the expression of functional RNAs, such as aptamers and antisense RNAs, both in basic research and in gene therapy.

tRNAVal-heterodimeric maxizymes with high potential as geneinactivating agents: simultaneous cleavage at two sites in HIV-1 Tat mRNA in cultured cells.

It has been demonstrated that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). Intracellular activities of heterodimeric maxizymes and conventional ribozymes, under the control of a human tRNAVal-promoter, were compared against the cleavage of HIV-1 tat mRNA. The pol III-driven maxizymes formed very active heterodimers, and they successfully cleaved HIV-1 tat mRNA in mammalian cells at two sites simultaneously. The cleaved fragments were identified directly by Northern blotting analysis. Despite the initial concerns that a complicated dimerization process and formation of inactive homodimers were involved in addition to the process of association with the target, the overall intracellular activities of tRNAVal-driven maxizymes were significantly higher in mammalian cells than those of two sets of independent, conventional hammerhead ribozymes that were targeted at the same two sites within HIV-1 tat mRNA. Because the tRNAVal-driven maxizymes tested to date have been more effective than tRNAVal-driven "standard" hammerhead ribozymes, the tRNAVal-driven heterodimeric maxizymes appear to have potential utility as gene-inactivating agents.

Factors governing the activity in vivo of ribozymes transcribed by RNA polymerase III.

In order to determine the parameters that govern the activity of a ribozyme in vivo, we made a systematic analysis of chimeric tRNAVal ribozymes by measuring their cleavage activities in vitro as well as the steady-state levels of transcripts, the half-lives of transcribed tRNAVal ribozymes, and their activities in both HeLa and H9 cells. These analyses were conducted by the use of transient expression systems in HeLa cells and stable transformants that express ribozymes. Localization of transcripts appeared to be determined by the higher-order structure of each transcribed tRNAVal ribozyme. Since colocalization of the ribozyme with its target RNA is important for strong activity of the ribozyme in vivo, the best system for tRNA-based expression seems to be one in which the structure of the transcript is different from that of the natural tRNA precursor so that processing of the tRNAVal ribozyme can be avoided. At the same time, the structure of the transcript must be similar enough to allow recognition, probably by an export receptor, so that the transcript can be exported to the cytoplasm to ensure colocalization with its target. In the case of several tRNAVal ribozymes that we constructed, inspection of computer-predicted secondary structures enabled us to control the export of transcripts. We found that only a ribozyme that was transcribed at a high level and that had a sufficiently long half-life, within cells, had significant activity when used to withstand a challenge by human immunodeficiency virus type 1.

Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

A simple assay system for examination of the inhibitory potential in vivo of decoy RNAs, ribozymes and other drugs by measuring the Tat-mediated transcription of a fusion gene composed of the long terminal repeat of HIV-1 and a gene for luciferase.

Nucleic acid-based drugs, including antisense RNA and DNA, ribozymes and decoys appear to have potential for the suppression of the expression of specific genes. To allow the examination of the potential of such agents in vivo as anti-HIV drugs in standard laboratories, where facilities for handling live virions are not available, we constructed a simple assay system (HIV-1 model) that allows measurement of the extent of inhibition of Tat-mediated transcription of HIV-1 by nucleic acid-based drugs and other agents. In cells that harbor a stable chimeric long terminal repeat (LTR)-Luc construct (a fusion gene consisting of the LTR of HIV-1 and the gene for luciferase), total luciferase activity in an aliquot of cell lysate is dose- and promoter-dependent on transfection with a Tat expression plasmid, reflecting the character of the LTR promoter of HIV. When HeLa cells were co-transfected with the Tat expression plasmid and another plasmid that encoded the U6 promoter or the promoter of the gene for tRNA(Val) linked to the trans-activating response (TAR) sequence, total luciferase activity was inhibited by 60 or 40%, respectively. The inhibition was also dependent on the dose of the TAR expression plasmid. These results demonstrate the usefulness of this simple assay system for detection of the efficacy of a decoy RNA or a ribozyme in vivo, without a requirement for HIV-infected cells, by measurement of luciferase activity in vitro.

Ribozyme targeting of receptor for advanced glycation end products in mouse mesangial cells.

Accumulation of extracellular matrix is a characteristic of diabetic nephropathy, and advanced glycation end products (AGEs) are considered to play an important role in the mechanism. To investigate the involvement of the receptor for AGE (RAGE) in upregulation of type IV collagen by AGEs, we applied the hammerhead ribozyme for targeting RAGE. We established a stable mouse mesangial cell line that produces the RAGE-specific ribozyme (Rz-RAGE). Both the RAGE mRNA and protein were decreased in the cell line. The amount of type IV collagen mRNA increased by AGEs' treatment in control cells. In contrast, the increase of type IV collagen induced by AGEs was not observed in the Rz-RAGE-producing cells. We conclude that the induction of type IV collagen by AGEs is mediated by RAGE and this mechanism could be involved in diabetic nephropathy. This study also suggested the experimental/therapeutic potential of hammerhead ribozymes.

Imbalance between serum matrix metalloproteinase-2 and its inhibitor as a predictor of recurrence of urothelial cancer.

Serum levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) were evaluated as prognostic indicators of the recurrence of urothelial cancer. Sera were obtained from 127 healthy control subjects and 97 urothelial cancer patients who underwent complete resection and were measured for MMP-2 and TIMP-2 using a one-step enzyme immunoassay. The relationship between the serum MMP-2/TIMP-2 ratio and the recurrence of urothelial cancer was examined. The mean serum MMP-2/TIMP-2 ratio in the 31 advanced urothelial cancer patients with recurrence was significantly higher than that in the 22 patients without recurrence (P = 0.0029) and in the 44 superficial bladder cancer patients (P < 0.0001). The 1- and 3-year disease-free survival rates in the patients with high MMP-2/TIMP-2 ratios (50% and 12%) were significantly poorer than those of the patients with normal ratios (82% and 56%) (P = 0.0152). Univariate and multivariate analyses of recurrence demonstrated that the serum MMP-2/TIMP-2 ratio is a significant independent indicator of advanced urothelial cancer. Our results indicate that an imbalance between the serum levels of MMP-2 and TIMP-2 could be a new predictor of recurrence in advanced urothelial cancer patients.

Inhibition of transcription by the TAR RNA of HIV-1 in a nuclear extract of HeLa cells.

Regulation of transcription of human immunodeficiency virus type-1 (HIV-1) requires specific interaction of Tat protein with the trans-activation response region (TAR). Inhibition of replication of HIV-1 has previously been achieved with a TAR decoy, namely a short RNA oligonucleotide that corresponded to the sequence of the authentic TAR RNA. Since TAR RNA has the potential to interact with cellular factors, we examined the effect of TAR RNA on efficiency of transcription in nuclear of HeLa cell extracts. We performed an in vitro transcription assay in the presence of authentic TAR RNA using a template that was driven by the CMV (cytomegalovirus) early promoter in a HeLa nuclear extract and found, for the first time, that TAR RNA inhibited transcription by approximately 60-70% independently of the Tat-TAR interaction. Furthermore, we evaluated inhibition of transcription by variants of TAR RNA and found that the TAR RNA loop, bases surrounding the loop, the triple base bulge and the 'lower' stem region of TAR RNA were responsible for the inhibition of transcription. Taken together, earlier reports on proteins that bind to TAR RNA and the present results suggest that integrity of TAR RNA is important for efficient binding to cellular transcription factors. As judged from the significant inhibition observed in this study, the TAR decoy might sequester transcription factors and thus it might potentially be able to inhibit transcription of housekeeping genes that are unrelated to Tat function.

Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli.

For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.). As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo. If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.

Activities of tRNA-embedded dimeric minizymes.

Ribozymes have been shown to be potent inhibitors of gene expression and viral function. Effects of ribozyme-mediated repression to target gene in living cells are correlated with the amounts of expression and stabilities of ribozyme molecules. In our previous study, it was demonstrated that a minimized hammerhead ribozyme, minizyme, with high activity forms a dimeric structure with a common stem II. We constructed dimeric minizymes that could cleave the BCR-ABL chimeric (b2a2) mRNA which had been difficult target for conventional hammerhead ribozymes without damaging the normal ABL mRNA. In order to achieve high expression of these dimeric minizymes in vivo for the treatment of CML, we embedded the dimeric minizyme portion downstream of a tRNA(Val) promoter sequence which could be recognized by RNA polymerase III. We determined cleavage activities of tRNA-embedded dimeric minizymes and compared the activities between tRNA-embedded hammerhead ribozyme and tRNA-embedded dimeric minizymes. All tRNA-embedded dimeric minizymes tested were capable of cleavage the target substrate. The activity of the tRNA-embedded dimeric minizyme targeted at BCR-ABL mRNA was almost the same as that of the naked dimeric minizymes. Interestingly, the cleavage activity of tRNA-embedded dimeric minizymes was higher than that of tRNA-embedded conventional hammerhead ribozyme.

Elevation of serum levels of matrix metalloproteinase-2 and -3 as new predictors of recurrence in patients with urothelial carcinoma.

BACKGROUND: The relationship between serum levels of matrix metalloproteinase-2 (MMP-2) and MMP-3 and recurrence in patients with urothelial carcinoma after complete resection was studied to determine whether the enzymes could be a new predictor of recurrence. METHODS: Serum levels of MMP-2 and MMP-3 in 146 healthy controls, 52 patients with superficial (noninvasive) and 35 patients with advanced (invasive or metastatic) bladder carcinoma, and 30 patients with advanced upper urothelial carcinoma (renal pelvis and ureter) were measured by a one-step sandwich enzyme assay. RESULTS: Among the 53 patients with advanced urothelial carcinoma who underwent complete resection, the 1- and 3-year disease free survival rates of patients with elevated serum levels of either or both of the enzymes were 51% and 19%, respectively, whereas those of patients with normal serum levels of these enzymes were much higher, 86% and 79%, respectively (P < 0.005). In pT2N0M0 and pT3N0M0 patients who underwent complete resection, the recurrence rate in those with preoperative elevated serum levels of either or both of the enzymes was significantly higher than that in patients with normal levels (75% vs. 8%; P < 0.001). The 1- and 3-year disease free survival rates of pT2N0M0 and pT3N0M0 patients with elevated serum levels of either or both of the enzymes were 55% and 21%, respectively, which was significantly lower than that of patients with normal levels (92%; P < 0.001). CONCLUSIONS: The results of this study indicate that the elevation of serum levels of either MMP-2 or MMP-3 or both could be new predictors of recurrence in patients with advanced urothelial carcinoma after complete resection.

Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase.

The cellular 300 kDa protein known as p300 is a target for the adenoviral E1A oncoprotein and it is thought to participate in prevention of the G0/G1 transition during the cell cycle, in activation of certain enhancers and in the stimulation of differentiation pathways. In order to determine the exact function of p300, as a first step we constructed a simple assay system for the selection of a potential target site of a hammerhead ribozyme in vivo. For the detection of ribozyme-mediated cleavage, we used a fusion gene (p300-luc) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene. We were also interested in the correlation of the GUX rule, for the triplet adjacent to the cleavage site, with ribozyme activity in vivo. Therefore, we selected five target sites that all included GUX The rank order of activities in vitro indeed followed the GUX rule; with respect to the kcat, a C residue as the third base (X) was the best, next came an A residue and a U residue was the worst (GUC > GUA > GUU). However, in vivo the tRNA(Val) promoter-driven ribozyme, targeted to a GUA located upstream of the initiation codon, had the highest inhibitory effect (96%) in HeLa S3 cells when the molar ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4. Since the rank order of activities in vivo did not conform to the GUX rule, it is unlikely that the rate limiting step for cleavage of the p300-luc mRNA was the chemical step. This kind of ribozyme expression system should be extremely useful for elucidation of the function of p300 in vivo.

Characterization of several kinds of dimer minizyme: simultaneous cleavage at two sites in HIV-1 tat mRNA by dimer minizymes.

A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem-loop II. In a previous study we demonstrated that a minizyme with high activity forms a dimeric structure with a common stem II. Because of their dimeric structure, minizymes are potentially capable of cleaving a substrate at two different sites simultaneously. In order to examine the properties of different kinds of minizyme, we constructed a number of minizymes with short oligonucleotide linkers (2-5 bases) instead of stem-loop II and examined their cleavage activities against HIV-1 tat mRNA. Analyses of melting curves, as well as Arrhenius plots, revealed that, in general, the longer the oligonucleotide linkers, the more stable and more active were the dimer minizymes. All minizymes examined cleaved the target substrate at two sites simultaneously. The activity of the dimer minizyme with a 5 nt linker was higher than that of the parental hammerhead ribozyme because the latter full-sized ribozyme was able to cleave at one site only.

[Measurement of serum tissue polypeptide antigen (TPA) in patients with malignant tumor using prolifigen TPA-M "Daiichi" kit].

Serum tissue polypeptide antigen (TPA) was measured using a newly developed Prolifigen TPA-M "Daiichi" kit in 1,236 healthy subjects, 2,867 patients with malignant tumors, and 901 with benign diseases. Because 94.0% of healthy subjects had serum TPA under 70 U/l, the cut-off value was set at 70 U/l. Serum TPA was elevated in more than 50% of patients with head and neck cancer, lung cancer, liver cancer, gallbladder or bile duct cancer, pancreatic cancer, colorectal cancer, ovarian cancer, and prostate cancer. The overall positive rate in malignant tumors was 55.5%. Serum TPA was higher in advanced cancer than in earlier stage cancer, and decreased after the resection of the tumor. The false positive rate in benign diseases was 31.3%. ROC analysis revealed the usefulness of TPA as a tumor marker in many cancers. The correlation coefficient between TPA and CYFRA 21-1, and between TPA and TPSA, was 0.747 and 0.694, respectively. In conclusion, measurement of serum TPA using the new kit is useful in the management of patients with various malignant tumors.

Ribozymes: from mechanistic studies to applications in vivo.

The hammerhead ribozyme belongs to the class of molecules known as antisense RNAs. However, because of short extra sequences that form the so-called catalytic loop, it can act as an enzyme. Since the catalytic domain captures magnesium ions and magnesium ions can cleave phosphodiester bonds, hammerhead ribozymes are recognized as metalloenzymes. In general, the cleavage of phosphodiester bonds involves acid/base catalysis, with proton transfer occurring in the transition state. When the possibility of such a proton-transfer process was examined by measuring solvent isotope effects, it became apparent that no proton transfer occurs in the transition state during reactions catalyzed by a hammerhead ribozyme. It is likely, therefore, that hammerhead ribozymes exploit the general double-metal-ion mechanism of catalysis, with Mg2+ ions coordinating directly with the attacking and leaving oxygen moieties. Since the hammerhead ribozyme is one of the smallest RNA enzymes known and has potential as an antiviral agent, thus ribozyme has been extensively investigated for applications in vivo. Ribozymes are described that have possible utility as agents against HIV-1.