-
[show abstract]
[hide abstract]
ABSTRACT: We previously developed a promoter that was responsive to radiation by randomly combining cis-elements of transcription factors that are activated in response to radiation in prostate cancer cells. The promoter enhanced the expression of the luciferase gene linked downstream by more than 10-fold 12 h after X-ray irradiation at 10 Gy. However, without radiation, it still significantly drove its expression. To suppress expression while retaining its enhancement in response to radiation, we focused our attention on microRNAs (miRNAs). miRNAs are a group of non-coding RNAs approximately 22 nucleotides long that control gene expression by binding to a target sequence residing on the 3'-untranslated region (3'UTR) of a target gene. We identified 8 miRNAs that were downregulated in response to X-ray irradiation, and inserted artificial target sequences composed of randomly combined complementary sequences into 3 representative miRNAs into the 3'UTR of the luciferase gene. The target sequences suppressed the expression, and released the expression, after X-ray irradiation, as expected. When we combined an artificial target sequence with the radiation-responsive promoter, it resulted in a clear-cut gene regulation of expression that was greater than that induced by the promoter alone.
International Journal of Molecular Medicine 04/2013; · 1.98 Impact Factor
-
Ryohei Ogawa,
Akihiro Morii,
Akihiko Watanabe,
Zheng-Guo Cui,
Go Kagiya,
Shigekazu Fukuda,
Kyo Kume,
Takashi Hasegawa,
Masanori Hatashita,
Hironori Izumi,
Tetsuya Ishimoto,
Loreto B Feril
[show abstract]
[hide abstract]
ABSTRACT: Radio-genetic therapy is a combination of radiation therapy and gene therapy that may solve some of the problems associated with conventional radiotherapy. A promoter responsive to radiation was obtained from a promoter library composed of DNA fragments created by linking the TATA box signal to randomly combined binding sequences of transcription factors that are reactive to radiation. Each promoter connected to the luciferase gene, was evaluated by luciferase expression enhancement in transfected cells after X-ray irradiation. The reactivity of the best promoter was improved by the random introduction of point mutations and the resultant promoter showed more than a 20-fold enhancement of the luciferase expression after X-ray irradiation at 10 Gy. The expression of downstream genes was also enhanced in stably transfected cells not only by X-rays but also by proton beam irradiation; and either enhancement was attenuated when an anti-oxidant was added, thus suggesting the involvement of oxidative stress in the promoter activation. Constructed promoters were also activated in tumors grown in mice. In addition, cell killing with the fcy::fur gene (a suicide gene converting 5-fluorocytosin to highly toxic 5-fluorouracil) increased dose-dependently with 5-fluorocytosin only after X-ray irradiation in vitro. These results suggest that promoters obtained through this method could be used for possible clinical applications.
Bioengineered. 08/2012; 4(1).
-
[show abstract]
[hide abstract]
ABSTRACT: We chose promoters responsive to sonication in LNCap cells, a prostate cancer cell line, out of a library composed of DNA fragments constructed by linking the TATA box sequence to randomly combined cis-acting elements of transcription factors activated in response to radiation in prostate cancer cells. When a plasmid containing the luciferase gene under control of a promoter was transfected into LNCap cells and sonicated with 1MHz ultrasound at 0.5W/cm(2), 10% DF for 60s, 13 promoters showed more than 10-fold enhancement compared with their counterparts without sonication 12h after sonication. As to their responsiveness to sonication, the best two promoters were then compared to clone 880-8, a derivative from clone 880 that was created by random introduction of point mutations and was shown to have an improved response to X-ray irradiation. We then took clone 880-8 for further analyses since it showed the highest enhancement to sonication, though not statistically significant from the others. Next, we employed a retrovirus vector and stably introduced the luciferase gene under control of clone 880-8 into LNCap cells to establish a cell line. When the cell line was sonicated with 1MHz ultrasound at 0.5W/cm(2), 10% DF for 60s, luciferase expression was enhanced up to 14.8-fold 12h after sonication. We then established another cell line by replacing the luciferase gene with the fcy::fur gene, a suicide gene, and when the cell line was sonicated with 1MHz ultrasound at 0.5W/cm(2), 10% DF for 60s, expression of the gene was enhanced, showing the maximum expression 12-24h after sonication. When the cells were incubated in medium containing 5-fluorocytosine, cell survival ratio decreased dose dependently with 5-fluorocytosine only after sonication treatment, suggesting this promoter could be utilized for gene expression control with ultrasound.
Ultrasonics Sonochemistry 05/2012; 20(1):460-7. · 3.57 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Part one of this review focused on the thermal and mechanical effects of low-intensity ultrasound (US). In this second and
final part of the review, we will focus on and discuss various aspects of low-intensity US, with emphasis on the biomolecular
effects, US-mediated gene transfection (sonotransfection), and US-mediated permeabilization (sonopermeabilization). Sonotransfection
of different cell lines in vitro and target tissues in vivo have been reported. Optimization experiments have been done and
different mechanisms investigated. It has also been found that several genes can be up-regulated or down-regulated by sonication.
As to the potential therapeutic applications, systemic or local sonotransfection might also be a safe and effective gene therapy
method in effecting the cure of local and systemic disorders. Gene regulation of target cells may be utilized in modifying
cellular response to a treatment, such as increasing the sensitivity of diseased cells while making normal cells resistant
to the side effects of a treatment. Advances in sonodynamic therapy and drug sonopermeabilization also offer an ever-increasing
array of therapeutic options for low-intensity US.
Journal of Medical Ultrasonics 04/2012; 35(4):161-167. · 0.33 Impact Factor
-
Takashi Kondo,
Toru Yoshida, Ryohei Ogawa,
Mariame A. Hassan,
Yukihiro Furusawa,
Qing-Li Zhao,
Akihiko Watanabe,
Akihiro Morii,
Loreto B. Feril,
Katsuro Tachibana,
Hiroshi Kitagawa,
Yoshiaki Tabuchi,
Ichiro Takasaki,
Mohammad H. Shehata,
Nobuki Kudo,
Kazuhiro Tsukada
[show abstract]
[hide abstract]
ABSTRACT: PurposeIn this study, the effects of low-intensity pulsed ultrasound (LIU) as an adjuvant to doxorubicin (DOX) treatment was further
investigated in comparison to hyperthermia as another widely used adjuvant. The effects were compared with respect to cell
killing and apoptosis induction in U937 cells. Human primary liver cancer (PLC) cells were also used to evaluate the effects
of the combinations. The use of an echo contrast agent was investigated for further enhancement of cytotoxicity. Finally,
the acoustic mechanisms involved were investigated.
MethodsThe effects of different treatment regimens on cell viability were determined using the Trypan blue dye-exclusion test. Apoptosis
induction was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. The mechanistic
study involved electron paramagnetic spin trapping for detecting free radical formation as an indicator of the occurrence
of inertial cavitation and spectrophotometry for sucrose hydrolysis as an indicator for noncavitational effects.
ResultsThe combination treatments exerted synergistic effects on cytotoxicity depending on the acoustic conditions used. The use
of LIU as an adjuvant to DOX treatment was shown to be superior to the use of hyperthermia as an adjuvant. Moreover, the combination
seems to be promising for other cancer types provided that the acoustic conditions are properly selected with respect to drug
concentration. The key ultrasound mechanism responsible for the synergism observed was shown to be the production of free
radicals by inertial cavitation. Non-cavitational forces were also shown to contribute to the effect.
ConclusionThis study is motivating to engage in in vivo research with various cancer types as a step toward clinical applicability and
is emphasizing on the importance of developing therapeutic protocols for setting LIU parameters with respect to other therapeutic
conditions.
Journal of Medical Ultrasonics 04/2012; 36(2):61-68. · 0.33 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: PurposeTo develop artificial promoters that are activated in response to sonication and to determine these properties in vitro.
MethodsThe binding sites of four transcription factors (nuclear factor-kappa B, activating protein-1, nuclear factor-Y, and CArG
element binding factor A) that are activated by oxidative stress were randomly ligated and linked to a TATA-box sequence to
control the luciferase gene located downstream. Transiently transfected HeLa cells from human cervical cancer with a plasmid
vector containing such a gene cassette were exposed to sonication, and enhancement of luciferase expression was assessed by
dual luciferase assay.
ResultsOf 62 promoters constructed, two promoters, designated clone 31 and clone 62 promoters, showed a more than tenfold enhancement
6 h after sonication with 1-MHz ultrasound at 1.0 W/cm2 for 60 s. These promoters were activated in a dose-dependent manner with the intensity and duration of sonication. The activation
was attenuated by addition of dimethyl sulfoxide, an antioxidant, suggesting that oxidative stress was involved. The clone
31 promoter responded to each of two serial sonications. When sonicated 24 h after the first sonication, the peak of promoter
enhancement was higher than that after the first sonication.
ConclusionsA promoter sensitively responsive to sonication was constructed using the above method, possibly leading to the construction
of a promoter of interest that could be applied for clinical use.
Journal of Medical Ultrasonics 04/2012; 36(1):9-17. · 0.33 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: IntroductionIt has been shown that killing of suspended cells by low-intensity ultrasound (0.08–0.11W/cm2) can be enhanced by a mild non-lethal hypotonic (146 mOsm) medium.
PurposeIn this study we wished to determine whether hypotonia-induced cell swelling of suspension cells was directly related to enhancement
of ultrasound-mediated cell killing, and to verify whether similar effects could be observed on circulating and attached cells.
MethodsU937 cells under mild hypotonia were exposed to ultrasound for different times with real-time monitoring of cell size using
a particle-size-distribution analyzer. To study the effect on attached cells, HeLa cells were exposed to ultrasound while
under hypotonia in an in vivo-simulated set-up.
ResultsThe result showed that the enhanced cell killing (up to more than twice) was directly proportional to hypotonia-induced cell
swelling. Similar membrane damage based on PI staining could be observed on HeLa cells treated with hypotonia. An in vivo-simulated
circulating system also showed similar findings for hypotonia-enhanced ultrasound cell killing.
ConclusionThese findings showed that mild hypotonia can be used to augment the effect of ultrasound in the treatment of cancers, particularly
leukemia. The results showing that such enhancement is related to cell swelling could guide us toward proper timing of sonication
while under hypotonic treatment.
Journal of Medical Ultrasonics 04/2012; 37(1):3-8. · 0.33 Impact Factor
-
Ryohei Ogawa,
Akihiro Morii,
Akihiko Watanabe,
Zheng-Guo Cui,
Go Kagiya,
Shigekazu Fukuda,
Kyo Kume,
Takashi Hasegawa,
Masanori Hatashita,
Hironori Izumi,
Tetsuya Ishimoto,
Loreto B Feril
[show abstract]
[hide abstract]
ABSTRACT: We previously obtained an X-ray responsive promoter from 11 promoters that we constructed. In the present study, we aimed to determine the efficiency of our promoter construction method. In addition, the reactivity of the promoter to X-rays in vivo is also investigated.
Promoters constructed by linking the TATA box to randomly combined binding sequences of transcription factors activated by radiation were cloned to prepare a promoter library. Combinations of promoters and various genes were stably-transfected into HeLa cells to establish recombinant cell lines, which were then exposed to X-rays or a proton beam to observe gene expression enhancement with or without anti-oxidants. Tumors of luciferase-expressing recombinant cells on mice were exposed to X-rays and promoter activation was evaluated by detecting bioluminescence. As a model for in vitro suicide gene therapy, fcy::fur-expressing recombinant cells were exposed to X-rays before incubation with 5-fluorocytosin. Cell viability was determined with WST-8.
Twenty-five of the 62 promoters in the library enhanced luciferase activity over five-fold, 6 h after receiving 10 Gy of X-ray irradiation, suggesting the effectiveness of our method. Luciferase activity in recombinant cells was enhanced by X-rays and, to a lesser extent, by a proton beam. Anti-oxidants attenuated the enhancement, suggesting the involvement of oxidative stress. Promoters were less reactive to X-rays in tumors on mice. In our suicide gene therapy model, survival of post-irradiated cells decreased dose-dependently with 5-fluorocytosin.
Our method was efficient in generating radiation responsive promoters. Furthermore, we have successfully shown a potential therapeutic use for one of these promoters.
The Journal of Gene Medicine 03/2012; 14(5):316-27. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ultrasonic technologies pervade the medical field: as a long established imaging modality in clinical diagnostics; and, with the emergence of targeted high intensity focused ultrasound, as a means of thermally ablating tumours. In parallel, the potential of [non-thermal] intermediate intensity ultrasound as a minimally invasive therapy is also being rigorously assessed. Here, induction of apoptosis in cancer cells has been observed, although definitive identification of the underlying mechanism has thus far remained elusive. A likely candidate process has been suggested to involve sonochemical activity, where reactive oxygen species (ROS) mediate the generation of DNA single-strand breaks. Here however, we provide compelling new evidence that strongly supports a purely mechanical mechanism. Moreover, by a combination of specific assays (neutral comet tail and staining for γH2AX foci formation) we demonstrate for the first time that US exposure at even moderate intensities exhibits genotoxic potential, through its facility to generate DNA damage across multiple cancer lines. Notably, colocalization assays highlight that ionizing radiation and ultrasound have distinctly different signatures to their respective γH2AX foci formation patterns, likely reflecting the different stress distributions that initiated damage formation. Furthermore, parallel immuno-blotting suggests that DNA-PKcs have a preferential role in the repair of ultrasound-induced damage.
PLoS ONE 01/2012; 7(1):e29012. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We examined the molecular mechanisms involved in the adaptive response to cadmium (Cd)-induced apoptosis in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with 50 μM cadmium chloride (CdCl2) for 12 h, significant apoptosis occurred. This was associated with an increase in intracellular reactive oxygen species (ROS), sustained phosphorylation of JNK, activation of caspase-3, a decrease in Mcl-1 (anti-apoptotic Bcl-2 proteins), and increases in Bim, Noxa and tBid (a pro-apoptotic protein under the Bcl-2 family). No apoptosis occurred when the cells were treated with 1 μM CdCl2 for 72 h. However, pretreatment with low-dose CdCl2 dramatically altered the sensitivity of the cells to 50 μM CdCl2 with inhibition of apoptosis. Concomitantly, there were significant decreases in the generation of intracellular ROS and the activation of JNK. Pretreatment with 1 μM CdCl2 also attenuated the decrease in Mcl-1 and the increases in Bim, Noxa and tBid induced by 50 μM CdCl2. In conclusion, pretreatment with low-dose Cd inhibited apoptosis induced by high-dose Cd. The mechanism involves inhibition of intracellular ROS generation and JNK activation, and modulating the balance between the expression of Mcl-1 and its binding partners, Bim, Noxa and tBid.
Toxicology in Vitro 07/2011; 25(8):1687-93. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy gamma-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-kappaB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.
Journal of Bioscience and Bioengineering 07/2010; 110(1):118-23. · 1.79 Impact Factor
-
Takeshi Hori,
Takashi Kondo,
Masahiko Kanamori,
Yoshiaki Tabuchi, Ryohei Ogawa,
Qing-Li Zhao,
Kanwal Ahmed,
Taketoshi Yasuda,
Shoji Seki,
Kayo Suzuki,
Tomoatsu Kimura
[show abstract]
[hide abstract]
ABSTRACT: Despite improvements in chemotherapy and surgery in the treatment of osteosarcoma (OS), satisfactory results are still difficult to achieve. Novel therapeutic modalities need to be developed for osteosarcoma treatment. The combined effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ionizing radiation (IR) on human OS cells were investigated. IR and TRAIL treatment synergistically decreased the cell viability and enhanced apoptosis in OS cell lines. IR pretreatment enhances TRAIL-induced Bid and caspase-3 activations. Decreases in the expression levels of the antiapoptotic proteins c-FLIP and XIAP also associated with apoptosis enhancement. Furthermore, IR pretreatment enhanced DR4 and DR5 expressions at the transcription stage. These results can become the basic lines of evidence for the future treatment of OS using TRAIL with IR.
Journal of Orthopaedic Research 06/2010; 28(6):739-45. · 2.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The change in transfection efficiency of electroporation by the combined treatment with mild preheating (40 degrees C for 30 min) was investigated. HCT 116, HeLa S3 and SGC 7901 cells were treated with electroporation in medium containing pBKCMV-Luc plasmid with or without preheating. After 24 h, luciferase activity was increased by 36, 28 and 77%; luciferase mRNA transcription was increased by 45, 50 and 68%; and fluorescein isothiocyanate-dextran accumulation was increased by 9, 35 and 15% in preheated groups, respectively. These results demonstrate that the transfection efficiency was enhanced by mild preheating. The mechanism partially involves increased macromolecular particle accumulation.
Biotechnology Letters 11/2009; 32(3):367-71. · 1.68 Impact Factor
-
Takeshi Hori,
Takashi Kondo,
Masahiko Kanamori,
Yoshiaki Tabuchi, Ryohei Ogawa,
Qing-Li Zhao,
Kanwal Ahmed,
Taketoshi Yasuda,
Shoji Seki,
Kayo Suzuki,
Tomoatsu Kimura
[show abstract]
[hide abstract]
ABSTRACT: MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit the p53-MDM2 interaction and activate p53 signaling. The expressions of DR4 and DR5, Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, are regulated by p53. In this study, the combined effects of nutlin-3 and TRAIL on apoptosis were investigated in HOS and HCT116 cells, which express wild-type p53. Nutlin-3 and TRAIL synergistically enhanced apoptosis owing to their intrinsic and extrinsic pathway signals, respectively. The increase in the Bid expression level and the decrease in the expression levels of anti-apoptotic proteins, c-FLIP and XIAP, were involved in this apoptosis enhancement. Furthermore, nutlin-3 activated the DR5 promoter and increased the expression levels of DR5 at mRNA and protein levels. These results indicate that the combination, treated with nutlin-3 and TRAIL, is useful for apoptosis induction in malignant cells expressing wild-type p53.
Cancer letters 08/2009; 287(1):98-108. · 4.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Low modulation frequencies from 0.5 to 100Hz were shown to alter the characteristics of the ultrasound field producing solution agitation (<5Hz; region of "ultrasound streaming" prevalence) or stagnancy (>5Hz; region of standing waves establishment) (Buldakov et al., Ultrason. Sonochem., 2009). In this study, the same conditions were used to depict the changes in exogenous DNA delivery in these regions. The luciferase expression data revealed that lower modulations were more capable of enhancing delivery at the expense of viability. On the contrary, the viability was conserved at higher modulations whereas delivery was found to be null. Cavitational activity and acoustic streaming were the effecters beyond the observed pattern and delivery enhancement was shown to be mediated mainly through sonopermeation. To promote transfection, the addition of calcium ions or an echo contrast agent (Levovist((R))) was proposed. Depending on the mechanism involved in each approach, differential enhancement was observed in both regions and at the interim zone (5Hz). In both cases, enhancement in standing waves field was significant reaching 16.0 and 3.3 folds increase, respectively. Therefore, it is concluded that although the establishment of standing waves is not the only prerequisite for high transfection rates, yet, it is a key element in optimization when other factors such as proximity and cavitation are considered.
Journal of Controlled Release 08/2009; 141(1):70-6. · 5.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously found that the heme oxygenase-1 gene (hmox-1) was the most upregulated gene among 9,182 genes in human lymphoma U937 cells exposed to a 1-MHz continuous ultrasound using the cDNA microarray technique. However, little is known about the molecular mechanisms of the induction of hmox-1 expression by ultrasound. We investigated the mechanism using human prostate cancer DU145 cells in which expression of hmox-1 increased with sonication in a time and an intensity-dependent manner. When N-acetyl-L-cysteine or glutathione-monoethyl ester, a potent antioxidant, was added to cell culture, hmox-1 upregulation was attenuated, suggesting that oxidative stress caused by sonication is involved in this process. To identify cis-acting elements required for the ultrasound-mediated induction, we carried out transient expression assays with plasmids carrying the luciferase gene under control of deletion mutants of the 5'-flanking region of hmox-1. The results revealed that the upregulations by sonication were observed with deletion mutants carrying the E1 or E2 enhancer of the 5'-flanking region, suggesting stress-responsive elements (StRE) were involved in the induction because either enhancer contains a number of the element. Indeed, site-directed mutations within StRE decreased the reactivity of deletion mutants to sonication. A transcription factor NF-E2-related Factor 2 that binds to StRE would therefore be activated by oxidative stress induced by sonication.
Ultrasound in medicine & biology 10/2008; 35(1):155-64. · 2.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We compared the enhancement effects of three different echo contrast agents (ECAs); Levovist, YM454, and MRX-815H as artificial microbubbles on ultrasound mediated gene transfection (USMGT) with 1MHz ultrasound at 0.2MPa using a luciferase expression vector in PC3 cells and elucidated the mechanisms of differences of USMGT facilitation by these ECAs. At a concentration of each ECA that induced iso-survival, ECAs with lipid shell (YM454 and MRX-815H) facilitated USMGT higher than those without shell (Levovist), and the order of the ECAs facilitating free radical formation by sonication was; YM454>MRX-815H>Levovist. These results suggested that the lipid shell type ECAs facilitated gene transfer higher than that by the non-shell type ECA.
Cancer Letters 07/2008; 265(1):107-12. · 4.24 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Potential clinical use of ultrasound (US) in enhancing the effects of anticancer drugs in the treatment of cancers has been highlighted in previous reports. Increased uptake of drugs by the cancer cells due to US has been suggested as a mechanism. However, the precise mechanism of the enhancement has not yet been elucidated. Here, the combined effects of low-intensity pulsed US and doxorubicin (DOX) on cell killing and apoptosis induction of U937 cells, and mechanisms involved were investigated.
Human myelomonocytic lymphoma U937 cells were used for the experiments. Experiments were conducted in 4 groups: (1) non-treated, (2) DOX treated (DOX), (3) US treated (US), and (4) combined (DOX + US). In DOX +US, cells were exposed to 5 microM DOX for 30 min and sonicated by 1 MHz pulsed US (PRF 100 Hz, DF 10%) at intensities of 0.2-0.5 W/cm(2) for 60 s. The cells were washed and incubated for 6 h. The viability was evaluated by Trypan blue dye exclusion test and apoptosis and incorporation of DOX was assessed by flow cytometry. Involvement of sonoporation in molecular incorporation was evaluated using FITC-dextran, hydroxyl radical formation was measured by electron paramagnetic resonance-spin trapping, membrane alteration including lipid peroxidation and membrane fluidity by DOX was evaluated using cis-parinaric acid and perylene fluorescence polarization method, respectively.
Synergistic enhancement in cell killing and additive enhancement in induction of apoptosis were observed at and above 0.3 W/cm(2). No enhancement was observed at 0.2 W/cm(2) in cell killing and induction of apoptosis. Hydroxyl radicals formation was detected at and above 0.3 W/cm(2). The radicals were produced more in the DOX + US than US alone. Incorporation of DOX was increased 13% in DOX + US (vs. DOX) at 0.5 W/cm(2). Involvement of sonoporation for increase of drug uptake was suggested by experiment using FITC-labeled dextran. We made the hypothesis that DOX treatment made the cells weaken against the mechanical effect of the US. Although treatment of DOX at 5 microM for 30 min did not affect lipid peroxidation and fluidity of cell membrane significantly, higher concentration and longer treatment of DOX induced the significant alteration of cell membrane.
Mechanisms of enhancements could be (1) increase in incorporation of the DOX by US involved with sonoporation, (2) enhancement of the cavitation by DOX. Cavitation is required for the enhancement of the effect of DOX. Although the precise involvement of the membrane modifications by DOX in the enhancement remains to be elucidated, they could be involved in the latent effects.
Cancer Chemotherapy and Pharmacology 05/2008; 61(4):559-67. · 2.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A promoter that is activated by ionizing radiation may be a useful tool for cancer therapy since, with such a promoter, the therapeutic gene can be expressed only in cancer tissues by irradiation. An artificially constructed promoter is advantageous as natural promoters may have physiological limitations. However, reasonably designing a promoter is hampered by shortage of information about the relationship between the structure and properties of a promoter DNA.
Binding sites of four transcription factors that were activated by radiation were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected cancer cells with such a vector were exposed to X-ray irradiation and enhancement of luciferase expression was assessed. To improve promoter sensitivity, mutations were randomly introduced into a constructed promoter by error-prone polymerase chain reaction (epPCR).
Of the 11 promoters constructed, the clone 11 promoter (clone 11 + TATA-box) showed a 5-fold enhancement 6 h after the 10 Gy X-ray irradiation in HeLa cells. A mutant designated the clone 11-9-37 promoter generated through two steps of epPCR showed a sensitivity 4.8 times higher than the clone 11 promoter to the 10 Gy X-rays, showing 21.6-fold enhancement of luciferase expression. Clone 11 was composed of 16 cis-acting elements, and the clone 11-9-37 promoter carried six point mutations.
A sensitively responsive promoter to radiation could be constructed using this method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.
The Journal of Gene Medicine 04/2008; 10(3):316-24. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Synthetic oligonucleotides containing one of four kinds of cis-acting elements, binding sites for activating protein-1 (AP-1), nuclear factor kappaB (NF-kappaB), CArG binding factor A (CBF-A), and nuclear factor Y (NF-Y), were randomly ligated to construct DNA fragments. These fragments were inserted into the SalI site of a promoter probe vector; pGL3-TATASal, which is located immediately upstream of the TATA box sequence of the human heme oxygenase 1 gene and linked to the luciferase gene to construct 11 plasmid vectors. When these vectors were introduced into PC-3 cells of human prostate cancer, 6 out of the 11 transfectants showed a significantly higher luciferase activity than pGL3-TATASal. The two strongest promoters (clone 6 and clone 11) were investigated further Clone 6 turned out to be the strongest, showing a 3.0- and 8.4-fold activity in comparison to the two frequently used promoters--the cytomegalovirus (CMV) immediate early promoter and the simian virus 40 (SV40) early promoter respectively. Clone 11 was less active than clone 6, but still showed higher activity than the two promoters. When the plasmids were introduced into nine other cell lines, their activities varied but were still comparable to the two promoters. These results indicate that the method used here is simple and efficient for constructing strong promoters that are potentially useful for vectors in either gene therapy or recombinant vaccine.
BioTechniques 06/2007; 42(5):628-33. · 2.67 Impact Factor
