Cisplatin selects short forms of the mitochondrial DNA OriB variant (16184-16193 poly-cytosine tract), which confer resistance to cisplatin.

A number of alternations in mitochondrial DNA (mtDNA) have been reported in different types of cancers, and the role of mtDNA in cancer has been attracting increasing interest. In order to investigate the relationship between mtDNA alternations and chemosensitivity, we constructed cybrid (trans-mitochondrial hybrid) cell lines carrying a HeLa nucleus and the mtDNA of healthy individuals because of the presence of somatic alternations in the mtDNA of many cancer cells. After a treatment with 1.0 µg/mL cisplatin for 10 days, we isolated 100 cisplatin-resistant clones, 70 of which carried the shorter mtDNA OriB variant (16184-16193 poly-cytosine tract), which was located in the control region of mtDNA. Whole mtDNA sequencing of 10 clones revealed no additional alternations. Re-construction of the HeLa nucleus and mtDNA from cisplatin-resistant cells showed that cisplatin resistance was only acquired by mtDNA alternations in the control region, and not by possible alternation(s) in the nuclear genome.

Mild hypothermia enhanced the protective effect of protein therapy with transductive anti-death FNK protein using a rat focal transient cerebral ischemia model.

We previously reported that the protein transduction domain fused FNK (PTD-FNK) protein, which was derived from anti-apoptotic Bcl-xL protein and thereby gained higher anti-cell death activity, has a strong neuroprotective effect on rat focal brain ischemia models. The aim of this study was to investigate the effect of PTD-FNK protein and hypothermia combined therapy on cerebral infarction. Male SD rats were subjected to 120min middle cerebral artery occlusion (MCAO) with intraluminal thread. Rats were divided into 4 groups: 1) 37°C vehicle administration (37V); 2) 37°C PTD-FNK administration (37F); 3) 35°C vehicle administration (35V); and 4) 35°C PTD-FNK administration (35F). PTD-FNK protein was intravenously administered 60min after the induction of MCAO. Hypothermia (35°C) was applied during 120min MCAO. Rats were sacrificed 24h later; infarct volumes were measured, and Bax, Bcl-2, TUNEL and caspase-12 immunostaining was evaluated. There was significant infarct volume reduction in 37F, 35V, and 35F groups compared to 37V. There was also a significant difference between 37F and 35F. This suggests that hypothermia enhanced the effect of PTD-FNK. Similar results were found in neurological symptoms. Caspase-12 and TUNEL staining showed a significant difference between 37F and 35F; however, Bax and Bcl-2 staining failed to show a difference. In this study we showed an additive protective effect of hypothermia on PTD-FNK treatment, and immunohistological results showed that the protective mechanisms might involve the inhibition of apoptotic pathways through caspase-12, but not through Bcl-2.

Mitochondrial DNA alterations in colorectal cancer cell lines.

Somatic mutations of mitochondrial DNA (mtDNA) have been reported in different types of cancers and are suggested to play roles in metastasis, cancer development and response to anticancer agents. To predict potential roles of mtDNA alterations in colorectal cancer, we determined the entire mtDNA sequence of eleven human-derived colorectal cancer cell lines and compared with the revised Cambridge Reference Sequence to identify nucleotide alterations. Four homoplasmic and six heteroplasmic alterations were found to be novel. Among them, homoplasmic G6709A (MT-CO1) and G14804A (MT-CYB) alterations cause amino acid changes in the highly conserved residues. Heteroplasmic G1576A (MT-RNR1) and G2975A (MT-RNR2) alterations are expected to make the stem structure of mitochondrial ribosomal RNAs unstable. These nucleotide alterations are candidates that could play important roles in cancer.

Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress.

Mitochondria combine the production of energy with an efficient chain of reduction-oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.

Mutations in the mitochondrial genome confer resistance of cancer cells to anticancer drugs.

The majority of cancer cells harbor homoplasmic somatic mutations in the mitochondrial genome. We show here that mutations in mitochondrial DNA (mtDNA) are responsible for anticancer drug tolerance. We constructed several trans-mitochondrial hybrids (cybrids) with mtDNA derived from human pancreas cancer cell lines CFPAC-1 and CAPAN-2 as well as from healthy individuals. These cybrids contained the different mitochondrial genomes with the common nuclear background. We compared the mutant and wild-type cybrids for resistance against an apoptosis-inducing reagent and anticancer drugs by exposing the cybrids to staurosporine, 5-fluorouracil, and cisplatin in vitro, and found that all mutant cybrids were more resistant to the apoptosis-inducing and anticancer drugs than wild-type cybrids. Next, we transplanted mutant and wild-type cybrids into nude mice to generate tumors. Tumors derived from mutant cybrids were more resistant than those from wild-type cybrids in suppressing tumor growth and inducing massive apoptosis when 5-fluorouracil and cisplatin were administered. To confirm the tolerance of mutant cybrids to anticancer drugs, we transplanted a mixture of mutant and wild-type cybrids at a 1:1 ratio into nude mice and examined the effect by the drugs on the drift of the ratio of mutant and wild-type mtDNA. The mutant mtDNA showed better survival, indicating that mutant cybrids were more resistant to the anticancer drugs. Thus, we propose that mutations in the mitochondrial genome are potential targets for prognosis in the administration of anticancer drugs to cancer patients.

Molecular hydrogen alleviates nephrotoxicity induced by an anti-cancer drug cisplatin without compromising anti-tumor activity in mice.

PURPOSE: Cisplatin is a widely used anti-cancer drug in the treatment of a wide range of tumors; however, its application is limited by nephrotoxicity, which is affected by oxidative stress. We have reported that molecular hydrogen (H(2)) acts as an efficient antioxidant (Ohsawa et al. in Nat Med 13:688-694, 2007). Here we show that hydrogen efficiently mitigates the side effects of cisplatin by reducing oxidative stress. METHODS: Mice were administered cisplatin followed by inhaling hydrogen gas (1% H(2) in air). Furthermore, instead of inhaling hydrogen gas, we examined whether drinking water containing hydrogen (hydrogen water; 0.8 mM H(2) in water) is applicable by examining oxidative stress, mortality, and body-weight loss. Nephrotoxicity was assessed by morphological changes, serum creatinine and blood urea nitrogen (BUN) levels. RESULTS: Inhalation of hydrogen gas improved mortality and body-weight loss caused by cisplatin, and alleviated nephrotoxicity. Hydrogen was detected in blood when hydrogen water was placed in the stomach of a rat. Consuming hydrogen water ad libitum also reduced oxidative stress, mortality, and body-weight loss induced by cisplatin in mice. Hydrogen water improved metamorphosis accompanying decreased apoptosis in the kidney, and nephrotoxicity as assessed by serum creatinine and BUN levels. Despite its protective effects against cisplatin-induced toxicity, hydrogen did not impair anti-tumor activity of cisplatin against cancer cell lines in vitro and tumor-bearing mice in vivo. CONCLUSION: Hydrogen has potential for improving the quality of life of patients during chemotherapy by efficiently mitigating the side effects of cisplatin.

Transduction of anti-cell death protein FNK suppresses graft degeneration after autologous cylindrical osteochondral transplantation.

This study shows that artificial super antiapoptotic FNK protein fused with a protein transduction domain (PTD-FNK) maintains the quality of osteochondral transplant by preventing chondrocyte death. Cylindrical osteochondral grafts were obtained from enhanced green fluorescent protein (EGFP)-expressing transgenic rats, in which living chondrocytes express green fluorescence, and submerged into medium containing PTD-FNK, followed by transplantation into cartilage defects of wild-type rats by impact insertion simulating autologous transplantation. The tissues were histologically evaluated by hematoxylin-eosin and Safranin-O staining. At 1 week, chondrocyte alignment was normal in the PTD-FNK treatment group, whereas all grafts without PTD-FNK treatment showed mixed cluster cell distribution. At 4 weeks, all grafts with PTD-FNK treatment showed almost normal matrix, whereas two grafts without PTD-FNK treatment showed fibrocartilage. Notably, all grafts with PTD-FNK retained high intensity of Safranin-O staining, but all grafts without PTD-FNK largely lost Safranin-O staining. PTD-FNK significantly suppressed a decrease in the survival rate and the density of EGFP-positive cells at 1 and 2 weeks, and this tendency continued at 4 weeks. The results of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-nick end-labeling staining showed that PTD-FNK inhibited cell death, indicating that PTD-FNK protects chondrocyte death and suppresses graft degeneration.

Involvement of mitoKATP channel in protective mechanisms of cerebral ischemic tolerance.

Little work has been performed to determine roles of mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) in ischemic preconditioning (IPC) in brain. To investigate the role on cerebral IPC, we examined effect of 5-hydroxydecanoate (5-HD), a selective mitoK(ATP) blocker, and diazoxide (DZX), a selective mitoK(ATP) opener on various IPC models. An IPC model with gerbil: 2 min bilateral common carotid arteries occlusion (BLCO)+24 h recovery+5 min BLCO. 5-HD, DZX, vehicle was administered 30 min before 5 min BLCO. Seven days later, surviving CA1 neurons were counted. A focal IPC model with rat: 15 min middle cerebral artery occlusion (MCAO)+48 h recovery+90 min MCAO. Twenty-four hours before 90 min MCAO, 5-HD, DZX, or vehicle was administered. One day after 90 min MCAO, neurological symptoms and infarct volumes were evaluated. An in vitro IPC model with primary neuronal cultures: 8 min oxygen-glucose deprivation (OGD)+24 h recovery+70 min OGD. Thirty minutes before 70 min OGD, 5-HD or DZX were added. One day later, surviving neurons were counted. Mitochondrial membrane potential was also monitored. 5-HD significantly attenuated the protective effect of IPC in gerbil model, rat model, and in vitro OGD model. DZX significantly facilitated the protective effect of IPC in gerbil and rat model. The mitochondrial membranes were depolarized with IPC, and 5-HD treatment significantly reduced this effect. These results strongly suggest that mitoK(ATP) channel activation plays a key role in development of a protective mechanism of cerebral IPC.

Therapeutic benefits of intrathecal protein therapy in a mouse model of amyotrophic lateral sclerosis.

When fused with the protein transduction domain (PTD) derived from the human immunodeficiency virus TAT protein, proteins can cross the blood-brain barrier and cell membrane and transfer into several tissues, including the brain, making protein therapy feasible for various neurological disorders. We have constructed a powerful antiapoptotic modified Bcl-X(L) protein (originally constructed from Bcl-X(L)) fused with PTD derived from TAT (TAT-modified Bcl-X(L)), and, to examine its clinical effectiveness in a mouse model of familial amyotrophic lateral sclerosis (ALS), transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) bearing a G93A mutation were treated by intrathecal infusion of TAT-modified Bcl-X(L). We demonstrate that intrathecally infused TAT-fused protein was effectively transferred into spinal cord neurons, including motor neurons, and that intrathecal infusion of TAT-modified Bcl-X(L) delayed disease onset, prolonged survival, and improved motor performance. Histological studies show an attenuation of motor neuron loss and a decrease in the number of cleaved caspase 9-, cleaved caspase 3-, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the lumbar cords of TAT-modified Bcl-X(L)-treated G93A mice. Our results indicate that intrathecal protein therapy using a TAT-fused protein is an effective clinical tool for the treatment of ALS.

Imaging mitochondrial redox environment and oxidative stress using a redox-sensitive fluorescent protein.

Redox-sensitive green fluorescent protein (roGFP) is a fluorescent protein in which two cysteines are placed adjacently in the barrel structure. Disulfide formation (oxidation) increases the absorption at short wavelengths (410 nm) at the expense of absorption at longer wavelengths (490 nm). The fluorescence ratio indicates reduction/oxidation, i.e., the redox potential at specific cellular locations.

Combination therapy with transductive anti-death FNK protein and FK506 ameliorates brain damage with focal transient ischemia in rat.

Many practical therapies have been explored as clinical applications for ischemic cerebral infarction; however, most are still insufficient to treat acute stroke. We show here a potential combination therapy in a rat focal ischemic model to improve neurological symptoms as well as to reduce infarct volumes at the maximum level. We applied protein transduction technology using artificial anti-death Bcl-xl derivative with three amino acid-substitutions (Y22F, Q26N and R165K) (FNK) protein fused with a protein-transduction-domain peptide (PTD-FNK). When PTD-FNK was administrated 1 h after initiating ischemia followed by the administration of an immunosuppressant FK506 with a 30-min time lag, infarct volumes of the total brain and cortex were markedly reduced to 27% and 14%, respectively. This procedure not only reduced the infarct volume and edema, but also markedly improved neurological symptoms. The therapeutic effect continued for at least 1 week after ischemia. FK506 inhibited the transduction of PTD-FNK in vitro, which explains the requirement of a time lag for the administration of FK506. An additional in vitro experiment showed that PTD-FNK, when administered 30 min before FK506, gave the maximal protective effect by reducing the intracellular calcium concentration. We propose that this combination therapy would provide a synergistic protective effect by both drugs, reducing adverse the effects of FK506.

Prevention of chemotherapy-induced alopecia by the anti-death FNK protein.

Many anticancer drugs attack rapidly dividing cells, including not only malignant cells but also hair follicle cells, and induce alopecia. Chemotherapy-induced alopecia (CIA) is an emotionally distressing side effect of cancer chemotherapy. There is currently no useful preventive therapy for CIA. We have previously constructed anti-death rFNK protein from rat Bcl-x(L) by site-directed mutagenesis to strengthen cytoprotective activity. When fused to the protein transduction domain (PTD) of HIV/Tat, the fusion protein PTD (TAT)-rFNK successfully entered cells from the outside in vitro and in vivo to exhibit anti-death activity against apoptosis and necrosis. Here, we show that topical application of FNK protected against CIA in a newborn rat model. The protective activity against hair-loss was observed in 30-1000 nM TAT-rFNK administrative groups in a dose-dependent manner. Furthermore, a human version of FNK (hFNK) fused to other PTD peptides exhibited a protective ability. These results suggest that PTD-FNK possesses protective activity against CIA and is not restricted to a sequence of PTD peptides or species of FNK. Thus, PTD-FNK represents potential to develop a useful method for preventing CIA in cancer patients.

PTD-mediated delivery of anti-cell death proteins/peptides and therapeutic enzymes.

Millions of unnecessary cells are removed from our body everyday by apoptosis to ensure our survivals. Apoptosis is a highly coordinated process. Failure in apoptotic regulation results in disease. A large number of studies have demonstrated that accelerated apoptosis is involved in degenerative diseases, ischemic injuries, immunodeficiency and infertility. These studies have also revealed the molecular mechanisms of apoptosis signal transduction to provide therapeutic targets. On the other hand, protein transduction technology has been developed to deliver full-length proteins to various tissues including the brain. So far, many studies have shown that in vivo delivery of therapeutic proteins/peptides, including anti-apoptotic proteins, an anti-oxidant enzyme, a neuroprotectant, enzymes involved in purine or tyrosine metabolism, caspase inhibitors, c-Jun N-terminal kinase inhibitors and an NF-kappaB inhibitor, by protein transduction technology mitigates various diseases in animal models.

Anti-apoptotic PTD-FNK protein suppresses lipopolysaccharide-induced acute lung injury in rats.

The present study was aimed at clarifying the effects of an anti-apoptotic protein for modulating symptoms in acute lung injury (ALI). From Bcl-x(L), a Bcl-2 family member, we constructed an artificial protein (FNK) and fused it with the protein transduction domain (PTD) of the HIV/Tat protein (PTD-FNK) to facilitate its permeation into cells. ALI was induced by intratracheal infusion of lipopolysaccharide (LPS) into Sprague-Dawley male rats. PTD-FNK was injected into the peritoneal cavity of the animals either 2 h before, or 3 h or 6 h after LPS challenge. All rats were sacrificed 24 h after the last treatment. Cell differential ratios and albumin concentration were estimated in bronchoalveolar lavage fluid. We examined histological change, myeloperoxidase activity, TUNEL assay, caspase-3/caspase-3-like activity and immunohistochemical reaction for caspase 3 (active form). In animals with PTD-FNK treatment, the albumin leakage was significantly attenuated with protection of tissue damage. Also, the apoptosis of alveolar wall cells was reduced by PTD-FNK treatment, while a total cell number and the neutrophil ratio were not changed. Human umbilical vein endothelial cells (HUVEC) and cells of an alveolar epithelial cell line (A549) were exposed to LPS or TNF-alpha with or without PTD-FNK treatment in vitro. Cell survival rates examined by trypan-blue exclusion assay were increased by PTD-FNK treatment in a concentration-dependent manner. Thus, PTD-FNK could play a protective role in ALI by suppressing apoptosis of alveolar epithelial cells and capillary endothelial cells despite of some effect on neutrophil activity.

Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress.

We have recently showed that molecular hydrogen has great potential for selectively reducing cytotoxic reactive oxygen species, such as hydroxyl radicals, and that inhalation of hydrogen gas decreases cerebral infarction volume by reducing oxidative stress [I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, K.-I. Katsura, Y. Katayama, S. Asoh, S. Ohta, Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med., 13 (2007) 688-694]. Here we show that the inhalation of hydrogen gas is applicable for hepatic injury caused by ischemia/reperfusion, using mice. The portal triad to the left lobe and the left middle lobe of the liver were completely occluded for 90min, followed by reperfusion for 180min. Inhalation of hydrogen gas (1-4%) during the last 190min suppressed hepatic cell death, and reduced levels of serum alanine aminotransferase and hepatic malondialdehyde. In contrast, helium gas showed no protective effect, suggesting that the protective effect by hydrogen gas is specific. Thus, we propose that inhalation of hydrogen gas is a widely applicable method to reduce oxidative stress.

Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals.

Acute oxidative stress induced by ischemia-reperfusion or inflammation causes serious damage to tissues, and persistent oxidative stress is accepted as one of the causes of many common diseases including cancer. We show here that hydrogen (H(2)) has potential as an antioxidant in preventive and therapeutic applications. We induced acute oxidative stress in cultured cells by three independent methods. H(2) selectively reduced the hydroxyl radical, the most cytotoxic of reactive oxygen species (ROS), and effectively protected cells; however, H(2) did not react with other ROS, which possess physiological roles. We used an acute rat model in which oxidative stress damage was induced in the brain by focal ischemia and reperfusion. The inhalation of H(2) gas markedly suppressed brain injury by buffering the effects of oxidative stress. Thus H(2) can be used as an effective antioxidant therapy; owing to its ability to rapidly diffuse across membranes, it can reach and react with cytotoxic ROS and thus protect against oxidative damage.

Transduction of anti-cell death protein FNK protects isolated rat hearts from myocardial infarction induced by ischemia/reperfusion.

Artificial anti-cell death protein FNK, a Bcl-x(L) derivative with three amino acid-substitutions (Y22F, Q26N, and R165K) has enhanced anti-apoptotic and anti-necrotic activity and facilitates cell survival in many species and cell types. The objectives of this study were (i) to investigate whether the protein conjugated with a protein transduction domain (PTD-FNK) reduces myocardial infarct size and improves post-ischemic cardiac function in ischemic/reperfused rat hearts, and (ii) to understand the mechanism(s) by which PTD-FNK exerts a protective effect. Isolated rat hearts were subjected to 35-min global ischemia, followed by 120-min reperfusion using the Langendorff methods. PTD-FNK (a total of 30 microl) was injected intramuscularly into the anterior wall of the left ventricle either at 1 min after induction of global ischemia (group A) or at 30 min after induction of global ischemia (at 5 min before reperfusion) (group B). In group A, infarct size was significantly reduced from 47.8+/-6.8% in the control to 30.4+/-5.2, 28.7+/-3.8, and 30.4+/-6.8% with PTD-FNK at 5, 50, and 500 nmol/l, respectively (p<0.05). Temporal recovery of left ventricular developed pressure at 60 min and 120 min after reperfusion was significantly better in PTD-FNK (50 and 500 nmol/l)-treated groups than in the control (p<0.05). In contrast, PTD-FNK treatment had no effect on group B. Western blot analysis showed that PTD-FNK markedly inhibited procaspase-3 cleavage (activation of caspase-3) and reduced the number of nuclei stained by a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphoshate nick-end labeling (TUNEL) assay. These findings suggest that PTD-FNK reduces the volume of myocardial infarction with corresponding functional recovery, at least in part, through the suppression of myocardial apoptosis following ischemia/reperfusion.

A protein derived from the fusion of TAT peptide and FNK, a Bcl-x(L) derivative, prevents cochlear hair cell death from aminoglycoside ototoxicity in vivo.

We constructed a powerful artificial cytoprotective protein, FNK, from an antiapoptotic member of the BCL-2 family, Bcl-x(L). To test the efficacy of FNK in protecting cochlear hair cells (HCs) from aminoglycoside-induced cell death in vivo, we fused FNK with protein transduction domain, TAT, of the HIV/Tat protein to construct a fusion protein of TAT-FNK. We demonstrated that, after an intraperitoneal administration to guinea pigs, TAT-myc-FNK protein was diffusely distributed in the cochlea, most prominently in the HCs and supporting cells, followed by the spiral ganglion cells, 3 hr after the injection. We next demonstrated that TAT-FNK attenuated cochlear damage induced by an ototoxic combination of kanamycin sulfate (KM) and ethacrynic acid (EA) administered at 2 different dosages: 400 mg/kg KM + 50 mg/kg EA and 200 mg/kg KM + 40 mg/kg EA. TAT-FNK or vehicle was intraperitoneally injected from 3 hr before through 5 hr after inducing the ototoxic insults, 14 days after which auditory brainstem response (ABR) and HC loss were evaluated. In comparison with vehicle-administered controls, the TAT-FNK protein significantly attenuated ototoxic drug-induced ABR threshold shifts and the extent of HC death at either dosage. The TAT-FNK protein also significantly reduced the amount of cleaved poly-(ADP-ribose) polymerase-positive HCs 8 hr after the ototoxic insults compared with that in the vehicle-administered controls. These findings indicate that systemically administered TAT-FNK was successfully delivered to the guinea pig cochlea and effectively prevented apoptotic cell death of the cochlear HCs induced by KM and EA.

Protection of hepatic cells from apoptosis induced by ischemia/reperfusion injury by protein therapeutics.

AIM: Apoptosis is involved in hepatic ischemia/reperfusion injury. The protein FNK, constructed from an anti-apoptotic protein Bcl-x(L), exhibits the stronger anticell death activity. We evaluated the effect of FNK on apoptosis after hepatic ischemia and reperfusion, using FNK-overexpressing transgenic mice and the HIV/Tat protein-transduction-domain (PTD) that mediates the introduction of FNK into cells when fused with FNK (PTD-FNK). METHODS: Mice were given hepatic ischemic insult for 90 min followed by reperfusion for 3 h. FNK overexpression was determined by immunohistochemistry and Western blot. PTD-FNK was intraperitoneally injected into wild mice 3 h before the insult. Liver injury was determined by the caspase activation, DNA fragmentation, and hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP- digoxigenin nick-end labelling (TUNEL) stainings. RESULTS: In FNK-transgenic mice, FNK overexpression inhibited the activation of caspase 3/caspase 3-like activity and DNA fragmentation caused by the injury. In wild mice preinjected with PTD-FNK, PTD-FNK significantly inhibited the caspase activation and DNA fragmentation, reduced the area of liver vacuolization, and protected hepatic cells surrounding blood vessels, irrespective of central or portal veins, from the ischemia/reperfusion damage. CONCLUSIONS: FNK inhibits apoptotic death due to the ischemia/reperfusion injury. Our results provide the reasonable expectation of therapeutic protein PTD-FNK for clinical applications, such as transplantation, to protect against ischemia/reperfusion injury.

Transduction of the anti-apoptotic PTD-FNK protein improves the efficiency of transplantation of bone marrow mononuclear cells.

Since most transplanted cells rapidly die in an ischemic environment by hypoxia and hyponutrition, it is crucial to know how to protect transplanted cells for improving transplantation efficiency. We examined whether the transduction of an artificial anti-cell death protein (PTD-FNK) into bone marrow mononuclear cells (BM-MNCs) prevents cell death and improves the transplantation efficiency of BM-MNCs in ischemic regions. Rat bone marrow cells were prepared from the femur and tibia and cultured on dishes precoated with human fibronectin in the absence of serum. BM-MNCs transduced with PTD-FNK survived better than those without the protein (P<0.008) and retained the potential to differentiate into endothelial progenitor cells (EPCs), as judged by the uptake of an acetylated low-density lipoprotein and the ability to bind lectin. Next, we used a co-culture system comprising human umbilical vein endothelial cells (HUVECs) and fibroblasts to examine angiogenic potential. HUVECs pretreated with PTD-FNK survived and formed a blood-vessel-like structure better than untreated cells (P<0.001). When BM-MNCs expressing EGFP were transplanted into ischemic areas of a male rat ischemic hindlimb model, the cells pretreated with PTD-FNK were incorporated into blood vessel with a higher efficiency than the untreated BM-MNCs (P=0.03). BM-MNCs protected through transduction of PTD-FNK maintained their angiogenic potential. Thus, PTD-FNK improves the transplantation efficiency of BM-MNCs into ischemic regions.