Differential diagnosis of Plasmodium falciparum and Plasmodium vivax in mixed infection by colorimetric nanogold probes.

Malaria is an infectious disease reported mostly in the tropical region. The most severe human malaria is Plasmodium falciparum since it can cause cerebral malaria. Therefore, the presence of P. falciparum either in single or mixed infection needs accurate diagnosis. In some mixed infections, the presence of P. falciparum may be cryptic which cannot be detected by microscopic examination. The molecular diagnosis is required in these cases. Many methods based on amplification of malaria parasite genes have been developed but most of them need sophisticated instruments. Here, we created a colorimetric method using probe immobilized gold nanoparticles (AuNPs) to detect the malaria parasite gene. Color changes rely on salt-induced aggregation of AuNPs in the presence or absence of DNA hybridization. Color changes could be observed either by a naked eye or UV-vis spectrophotometer. By this approach, single infection by the most common malaria parasite, P. falciparum or P. vivax could be differentially identified. Mixed infection of these two malaria species could also be clearly diagnosed including cases of cryptic P. falciparum. The novel nanogold based molecular malaria diagnosis is sensitive, specific, rapid and cheap ($0.94). The prepared nanogold malaria probes are stable for up to 3 months indicating their filed application in remote areas.

Genotyping of α-thalassemias by the colorimetric nanogold probes.

BACKGROUND: The novel colorimetric nanogold probe was created to genotype subgroups of the mostly found α-thalassemias. They are α-thalassemia 1 (SEA and THAI deletion) and α-thalassemia 2 (3.7-kb and 4.2-kb deletion). METHODS: The genotyping was performed by two-steps hybridizations. First step was hybridization of target DNA with the nanogold mixed probes of either α-thalassemia 1 or α-thalassemia 2. No hybridization in both reactions showing blue color indicated absence of abnormal genes causing these α-thalassemias. Positive reaction showing either red or purple color was further analyzed in second hybridization with the nanogold single probe. Positive of α-thalassemia 1 was genotyped with the single probes of both SEA and THAI deletion while those of α-thalassemia 2 were genotyped with both 3.7-kb and 4.2-kb deletion. RESULTS: Genotypic potency of the nanogold mixed and single probes was evaluated using both known diagnosed and suspected clinical samples. The results by naked eye were consistence with those analyzed by standard agarose gel electrophoresis. CONCLUSIONS: Potency of the colorimetric nanogold α-thalassemia probes was accurate, precise, sensitive, specific, simple, cheap and field applicable. Color reaction was simply visualized by naked eye. This development is an example of colorimetric molecular diagnosis which can be applied in any genetic detection.

Silver quartz crystal microbalance for differential diagnosis of Plasmodium falciparum and Plasmodium vivax in single and mixed infection.

The most severe form of malaria is cerebral malaria caused by Plasmodium falciparum. Standard malaria diagnosis is Giemsa stained peripheral blood smear but false negative findings are always reported. Moreover, mixed infections are underestimated by routine microscopy. Many methods have been developed to overcome these disadvantages and the most specific and sensitive is molecular diagnosis. Specific malaria genes are amplified by polymerase chain reaction (PCR) and many post-PCR methods have been created. We developed a gold fabricated quartz crystal microbalance (QCM) as a post-PCR method of malaria diagnosis. In this work a cheaper silver fabricated QCM was developed to identify both single and mixed infection of P. falciparum and Plasmodium vivax. The biotinylated malaria probe was immobilized on silver surface via specific avidin-biotin interaction. The target DNA fragment of 18s rRNA gene was amplified and hybridized with a QCM immobilized probe. Mass changes due to DNA hybridization were indicated by changes of quartz resonance frequencies. Validation showed that malaria silver QCM had high diagnostic potency. Evaluation of suspected 67 febrile blood samples from malaria endemic area demonstrated that the malaria silver QCM could identify both false negative and misdiagnosis cases of routine microscopy. The analysis cost of malaria silver QCM was $1/sample and analysis time was 30 min after blood collection. The malaria silver QCM is stable at tropical temperature for up to 6 months. Thus, it can be transported to be used in a remote endemic area. Thus, the malaria silver QCM is accurate, precise, rapid, cheap, and field applicable.

Study designs to investigate Nox1 acceleration of neoplastic progression in immortalized human epithelial cells by selection of differentiation resistant cells.

To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines - FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells.

Molecular diagnosis of α-thalassemias by the colorimetric nanogold.

A new application of gold nanoparticles (AuNPs) as a colorimetric method for gene detection of α-thalassemia 1 (SEA deletion) is reported here for the first time. This technique is based on color changes from salt-induced aggregation of un-hybridized nanogold probes after hybridization with the target DNA. Specific DNA probes were synthesized, thiol modified and conjugated on the surface of AuNPs. The target DNA was amplified and hybridized with the AuNPs-immobilized probe. Salt solution (NaCl) was added to induce aggregation of the un-hybridized nanogold probes. The color changes were visualized either by the naked eye or by UV-vis spectrophotometry at 520 nm. By this nanogold colorimetric method samples carrying normal α-globin genes could be successfully identified from samples carrying α-globin genes causing α-thalassemia 1 (SEA deletion), either as a carrier or disease form. Results demonstrated that the new colorimetric nanogold method is a definite gene diagnosis of α-thalassemia. It is accurate, simple, rapid, specific, sensitive, and cost effective. It is also a promising point-of-care testing (POCT) method for thalassemias and other genetic disorders. The new colorimetric nanogold is a method of choice for areas where access to sophisticated molecular diagnosis is limited.

Biosensor as a molecular malaria differential diagnosis.

BACKGROUND: In malaria diagnosis, specific gene identification is required in cases with subclinical infection or cases with mixed infection. This study applied the biosensor technology based on quartz crystal microbalance (QCM) to differentially diagnose the most common and severe malaria, Plasmodium falciparum and Plasmodium vivax. METHOD: The QCM surface was immobilized with malaria biotinylated probe. Specific DNA fragments of malaria-infected blood were amplified. Hybridization between the amplified products and the immobilized probe resulted in quartz frequency shifts which were measured by an in-house frequency counter. Diagnostic potency and clinical application of the malaria QCM were evaluated. RESULT: The malaria QCM could differentially diagnose blood infected with P. falciparum from that infected with P. vivax (p-value<0.05). No cross reaction with human DNA indicated the QCM specificity. Clinical application was evaluated using 30 suspected samples. Twenty-seven samples showed consistent diagnosis of the QCM with microscopy and rapid diagnosis tests (RDTs). Three samples reported "no malaria found" by microscopy showed P. falciparum infection by both QCM and the RDTs. CONCLUSION: The malaria QCM was developed with high accuracy, specificity, sensitivity, stability and cost-effectiveness. It is field applicable in malaria endemic area and might be a promising point of care testing.

Low cost biosensor-based molecular differential diagnosis of α-thalassemia (Southeast Asia deletion).

BACKGROUND: Thalassemias are genetic hematologic diseases which the homozygous form of α-thalassemia can cause either death in utero or shortly after birth. It is necessary to accurately identify high-risk heterozygous couples. We developed a quartz crystal microbalance (QCM) to identify the abnormal gene causing the commonly found α-thalassemia1, [Southeast Asia (SEA) deletion]. This work is an improved method of our previous study by reducing both production cost and analysis time. METHODS: A silver electrode on the QCM surface was immobilized with a biotinylated probe. The α-globin gene fragment was amplified and hybridized with the probe. Hybridization was indicated by changes of quartz oscillation. Each drying step was improved by using an air pump for 30 min instead of the overnight air dry. The diagnostic potency of the silver QCM was evaluated using 70 suspected samples with microcytic hypochromic erythrocytes. RESULTS: The silver QCM could clearly identify samples with abnormal α-globin genes, either homozygous or heterozygous, from normal samples. Thirteen out of 70 blood samples were identified as carrier of α-thalassemia1 (SEA deletion). Results were consistent with the standard agarose gel electrophoresis. Using silver instead of gold QCM could reduce the production expense 10-fold. An air pump drying the QCM surface could reduce the analysis time from 3 days to 4 h. CONCLUSIONS: The silver thalassemic QCM was specific, sensitive, rapid, cheap and field applicable. It could be used as a one-step definite diagnosis of α-thalassemia1 (SEA deletion) with no need for the preliminary screening test.

Diagnosis and genotyping of Plasmodium falciparum by a DNA biosensor based on quartz crystal microbalance (QCM).

BACKGROUND: Malaria infection with Plasmodium falciparum is an important basic health problem in the tropical and sub-tropical countries. The standard diagnostic method is blood film examination to visualize parasite morphology. However, in cases of low parasitemia or mixed infection with very low cryptic species, microscopy is not sensitive enough. Therefore, molecular techniques have been widely employed. METHODS: A label-free DNA biosensor based on quartz crystal microbalance (QCM) to diagnose and genotype P. falciparum was developed. Avidin-biotin interaction was used to coat the specific biotinylated probe on the gold surface of QCM. The gene encoding merozoite surface protein 2 (msp2) was amplified and the PCR products were then cut with restriction enzyme (MwoI). Enzymatic cutting made the PCR products suitable for QCM development. Hybridization between probe and enzymatic cutting DNA fragments resulted in frequency changes of the QCM. RESULTS: The newly developed QCM was tested for its diagnosis ability using both malaria laboratory strains and clinical isolates. The biosensor was sensitive at the sub-nanogram level, specific for only P. falciparum detection, no cross-reaction with P. vivax, and stable at room temperature for up to 6 months. Selection of msp2 as a target gene and a geno-typing marker made the QCM potentially useful for falciparum diagnosis simultaneously with genotyping. Potency was tested by genotyping two allelic families of P. falciparum, FC27 and IC1, using malaria laboratory strains, K1 and 3D7, respectively. CONCLUSIONS: The dual function QCM was successfully developed with high sensitivity and specificity, and was cost-effective, stable and field adaptable.

p53 Protects lung cancer cells against metabolic stress.

The preferential use of aerobic glycolysis for energy production by cancer cells, a phenomenon known as the 'Warburg effect', is well recognized and is being considered for therapeutic applications. However, whether inhibition of glycolysis will be effective in all types of cancer is unclear. The current study shows that a glycolytic inhibitor, 2-deoxy-D-glucose (2DG), exhibits the cytotoxic effect on non-small cell lung cancer in a p53-dependent manner. 2DG significantly inhibits ATP production in p53-deficient lung cancer cells (H358) but not in p53-wt cells (A549). In contrast to p53-wt cells, p53-defective cells are unable to compensate for their need of energy via oxidative phosphorylation (OXPHOS) when glycolysis is inhibited. In the presence of p53, increased ROS from OXPHOS increases the expression of p53 target genes known to modulate metabolism, including synthesis of cytochrome c oxidase 2 (SCO2) and TP53-induced glycolysis and apoptosis regulator (TIGAR). Importantly, 2DG selectively induces the expression of the antioxidant enzymes manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx1) in a p53-dependent manner. The results demonstrate that the killing of cancer cells by the inhibitor of glycolysis is more efficient in cancer cells without functional p53 and that p53 protects against metabolic stress by up-regulation of oxidative phosphorylation and modulation of antioxidants.

Quartz crystal microbalance-based biosensor for the detection of α-thalassemia 1 (SEA deletion).

BACKGROUND: DNA piezoelectric biosensors have become a promising tool in molecular medicine since they do not require any label or staining. Here, a DNA piezoelectric biosensor based on a quartz crystal microbalance (QCM) was created to identify abnormal genes causing α-thalassemia 1 (SEA deletion). METHODS: The functionalized gold electrode of the quartz crystal was coated with avidin and the biotinylated DNA probe was attached. The target gene causing α-thalassemia 1 was amplified and hybridized with the immobilized probe. DNA hybridization was indicated by changes in the quartz resonance frequencies. Diagnostic ability of the new α-thalassemia 1 biosensor was validated using both known and unknown blood samples. Specificity was tested using samples of ß-thalassemia and α-thalassemia 2. Stability of the sensor was also evaluated. RESULTS: The new biosensor could clearly identify α-thalassemia 1 (SEA deletion), both carrier and disease states, from the normal genotype. Identification accuracy was compatible to the standard gel electrophoresis. It was specific only to α-thalassemia 1 since no cross reaction was found with ß-thalassemia and α-thalassemia 2. The sensor could be kept at room temperature up to 6 months with consistent identification accuracy. CONCLUSIONS: The label free QCM based biosensor was successfully developed to diagnose an abnormal human globin gene causing α-thalassemia 1 (SEA deletion). Its accuracy, specificity and sensitivity were comparable to the standard method. Its stable diagnostic potency up to 6 months implied its field application in thalassemic control program.

p53 is an important factor for the radiosensitization effect of 2-deoxy-D-glucose.

Metabolic change in cancer cells by preferential production of energy through glycolysis is a well-documented characteristic of cancer. However, whether inhibition of glycolysis will enhance the efficacy of radiation therapy is a matter of debate. In this study which uses lung cancer as the model, we demonstrate that the improvement of radiotherapy by 2-deoxy-D-glucose (2DG) is p53-dependent. Based on clonogenic survival data, we show that p53-deficient lung cancer cells (H358) are more sensitive to 2DG treatment when compared to p53 wild-type lung cancer cells (A549). The effective doses of 2DG at 0.5-surviving fraction of A549 and H358 are 17.25 and 4.61 mM, respectively. Importantly, 2DG exhibits a significant radiosensitization effect in A549 cells but not in H358 cells. Treatment with 2DG increases radiation-induced p53 protein levels in A549 cells. siRNA inhibition of p53 in A549 cells reduces the radiosensitization effect of 2DG. Furthermore, ectopic expression of wild-type p53 in H358 cells significantly enhances the radiosensitization effect of 2DG as determined by colony formation assay. In nude mice injected with A549 cells, treatment of 2DG enhances the efficacy of radiation therapy. Together, these results suggest that inhibition of glycolysis may only be beneficial for radiation therapy of cancer expressing wild-type p53.

Mitochondrial and nuclear p53 localization in cardiomyocytes: redox modulation by doxorubicin (Adriamycin)?

Reactive oxygen (ROS) and nitrogen species (RNS) generation have been proposed to be an important mechanism of doxorubicin (Adriamycin; ADR)-induced cardiotoxicity and cardiomyocyte apoptosis, processes that may be mediated by p53 protein. We note that ADR treatment resulted in increased levels of p53 protein in cardiomyocyte mitochondria and nuclei. Modulation of the cardiomyocyte redox state in genetically engineered mice by modulation of enzymes involved in metabolism of ROS/RNS, manganese superoxide dismutase (MnSOD), or inducible nitric oxide synthase (iNOS), or a combination of these, regulated levels of mitochondrial/nuclear p53 in cardiomyocytes after ADR administration. These observations led to the hypothesis that mitochondrial/nuclear p53 localization and function in the cardiomyocyte response to ADR may be regulated through redox-dependent mechanism(s).

Evidence for p53 as guardian of the cardiomyocyte mitochondrial genome following acute adriamycin treatment.

The present study is an initial analysis of whether p53 may function as guardian of the cardiomyocyte mitochondrial genome, with mitochondrial p53 localization proposed to be involved in both mitochondrial DNA (mtDNA) repair and apoptosis. Subcellular distribution, protein levels, and possible function(s) of p53 protein in the response of cardiomyocytes to adriamycin (ADR) were analyzed. Levels and subcellular localization of proteins were determined by Western blot and immunogold ultrastructural analysis techniques. Here we demonstrate that stress caused by ADR induced upregulation of p53 protein in cardiomyocyte mitochondria and nuclei between 3 and 24 hr. Increased expression of PUMA and Bax proteins, pro-apoptotic targets of p53, was documented following ADR treatment and was accompanied by increased levels of apoptotic markers, with elevation of cytosolic cytochrome c at 24 hr and subsequent caspase-3 cleavage at 3 days. Mitochondrial p53 levels correlated with mtDNA oxidative damage. Loss of p53 in knockout mouse heart resulted in a significant increase in mtDNA vulnerability to damage following ADR treatment. Our results suggest that mitochondrial p53 could participate in mtDNA repair as a first response to oxidative damage of cardiomyocyte mtDNA and demonstrate an increase of apoptotic markers as a result of mitochondrial/nuclear p53 localization.

NF-kappaB-associated MnSOD induction protects against beta-amyloid-induced neuronal apoptosis.

Expression of manganese superoxide dismutase (MnSOD), a nuclear-encoded mitochondrial primary antioxidant enzyme, is protective against various paradigms of oxidative stress-induced brain injury. We have shown previously that the presence of an intronic nuclear factor site, kappaB (NF-kappaB), in the MnSOD gene is essential for the induction of MnSOD by tumor necrosis factor alpha (TNF-alpha). However, whether activation of NF-kappaB is protective against oxidative stress-induced neuronal injury is unclear. In the present study, we demonstrate that TNF-alpha activates NF-kappaB activity in neuronal, SH-SY5Y, cells and preferentially enhances the binding of p50 and p65 to the promoter/enhancer regions of the MnSOD gene. Binding of NF-kappaB members to the MnSOD gene leads to the induction of MnSOD mRNA and protein levels. Consequently, induction of MnSOD by TNF-alpha primes neuronal cells to develop resistance against subsequent exposure to beta-amyloid and FeSO(4). Taken together, these results suggest that NF-kappaB might exert its protective function by induction of MnSOD leading to subsequent protection against oxidative stress-induced neuronal injury.

NF-κB-Associated MnSOD induction protects against ß-amyloid-induced neuronal apoptosis.

Expression of manganese superoxide dismutase (MnSOD), a nuclear-encoded mitochondrial primary antioxidant enzyme, is protective against various paradigms of oxidative stress-induced brain injury. We have shown previously that the presence of an intronic nuclear factor site, κB (NF-κB), in the MnSOD gene is essential for the induction of MnSOD by tumor necrosis factor α (TNF-α). However, whether activation of NF-κB is protective against oxidative stress-induced neuronal injury is unclear. In the present study, we demonstrate that TNF-α activates NF-κB activity in neuronal, SH-SY5Y, cells and preferentially enhances the binding of p50 and p65 to the promoter/enhancer regions of the MnSOD gene. Binding of NF-κB members to the MnSOD gene leads to the induction of MnSOD mRNA and protein levels. Consequently, induction of MnSOD by TNF-α primes neuronal cells to develop resistance against subsequent exposure to ß-amyloid and FeSO4. Taken together, these results suggest that NF-κB might exert its protective function by induction of MnSOD leading to subsequent protection against oxidative stress-induced neuronal injury.

Induction of manganese superoxide dismutase (MnSOD) mediates cardioprotective effect of tamoxifen (TAM).

Tamoxifen (TAM), a synthetic nonsteroidal antiestrogen effectively and widely used for breast cancer treatment, is known to have antioxidant and cardioprotective effects, but whether the beneficial cardiovascular effect of TAM is linked to its antioxidant effect is unknown. In this study, we investigated the effect of TAM on the levels of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, in cardiac tissues and cardiomyocytes. TAM treatment induced MnSOD expression in vitro and in vivo. Cardiomyocytes isolated from TAM-pretreated mice also had higher MnSOD levels and fewer apoptotic cells compared to cardiomyocytes from control mice after adriamycin (ADR) treatment. To further confirm the role of MnSOD in the protection against ADR in cardiomyocytes, we used cardiomyocytes isolated from MnSOD knock-out (MnSOD(+/-)), wild-type (NTg) and human MnSOD transgenic (TgH) mice. TUNEL assay indicated that the percentage of cells undergoing apoptosis after ADR treatment was significantly greater in MnSOD(+/-) than in NTg or TgH cardiomyocytes. 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay showed that basal level of mitochondrial function was lower in MnSOD(+/-) cardiomyocytes than in NTg or TgH, and that MnSOD(+/-) was more sensitive to ADR. ADR treatment increased caspase activity, which was significantly higher in MnSOD(+/-) than in NTg or TgH cardiomyocytes. These results suggested that TAM-induced MnSOD expression is at least, in part, contribute to the cardioprotective effects of TAM.

Manganese superoxide dismutase and inducible nitric oxide synthase modify early oxidative events in acute adriamycin-induced mitochondrial toxicity.

In the present study, we used genetically engineered B6C3 mice [mice overexpressing manganese superoxide dismutase (TgM(+/+)), mice in which inducible nitric oxide synthase had been inactivated (iNOSKO(-/-)), and crosses of these two genotypes] to study the role of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in the development of acute Adriamycin-induced cardiotoxicity. Both nontransgenic and genetically engineered mice were treated with 20 mg/kg Adriamycin and cardiac left ventricular tissues studied at 0, 3, 6, and 24 hours. Ultrastructural damage and levels of 4-hydroxy-2-nonenal (4HNE) protein adducts and 3-nitrotyrosine (3NT) were determined in cardiomyocytes using immunogold ultrastructural techniques. Our previous results showed that Adriamycin caused mitochondrial injury without significant nuclear or cytoplasmic damage at early time points. Interestingly, overexpression of MnSOD protected against acute mitochondrial injury, whereas deficiency in iNOS potentiated mitochondrial injury in comparison with levels of injury present in cardiomyocyte mitochondria of nontransgenic mice. In TgM(+/+) mice, there was a significant inverse correlation between mitochondrial injury and 4HNE/3NT levels at all time points analyzed, suggesting that reactive oxygen species/reactive nitrogen species damage products directly regulated acute Adriamycin-induced mitochondrial injury in these mice. The present studies are the first to directly quantify the effects of MnSOD and iNOS on mitochondrial injury during acute Adriamycin-induced cardiotoxicity and show extensive and specific patterns of posttranslational modifications of mitochondrial proteins following Adriamycin treatment.

Measuring allelic heterogeneity in Plasmodium falciparum by a heteroduplex tracking assay.

We developed a novel Plasmodium falciparum genotyping strategy based on the heteroduplex tracking assay (HTA) method commonly used to genotype viruses. Because it can detect both sequence and size polymorphisms, we hypothesized that HTA is more sensitive than current methods. To test this hypothesis, we compared the ability of HTA and a nested polymerase chain reaction (PCR) to detect genetic diversity in 17 Thai samples. The HTA detected more MSP1 sequence variants in eight isolates (47%), less sequence variants in three isolates (18%), and an equal number of sequence variants in six isolates (35%), suggesting that HTA is equal to or more sensitive than the nested PCR. This study is a proof of concept that HTA is a sensitive allelic discrimination method able to determine genetic diversity in P. falciparum and warrants its use in studies of antimalarial drug efficacy.

Oxidative damage precedes nitrative damage in adriamycin-induced cardiac mitochondrial injury.

The purpose of the present study was to determine if elevated reactive oxygen (ROS)/nitrogen species (RNS) reported to be present in adriamycin (ADR)-induced cardiotoxicity actually resulted in cardiomyocyte oxidative/nitrative damage, and to quantitatively determine the time course and subcellular localization of these postulated damage products using an in vivo approach. B6C3 mice were treated with a single dose of 20 mg/kg ADR. Ultrastructural damage and levels of 4-hydroxy-2-nonenal (4HNE)-protein adducts and 3-nitrotyrosine (3NT) were analyzed. Quantitative ultrastructural damage using computerized image techniques showed cardiomyocyte injury as early as 3 hours, with mitochondria being the most extensively and progressively injured subcellular organelle. Analysis of 4HNE protein adducts by immunogold electron microscopy showed appearance of 4HNE protein adducts in mitochondria as early as 3 hours, with a peak at 6 hours and subsequent decline at 24 hours. 3NT levels were significantly increased in all subcellular compartments at 6 hours and subsequently declined at 24 hours. Our data showed ADR induced 4HNE-protein adducts in mitochondria at the same time point as when mitochondrial injury initially appeared. These results document for the first time in vivo that mitochondrial oxidative damage precedes nitrative damage. The progressive nature of mitochondrial injury suggests that mitochondria, not other subcellular organelles, are the major site of intracellular injury.

Human gingival mucosal keratinocytes exhibiting anchorage-independent growth express increased inducible nitric oxide synthase: regulation by MAP kinases.

Inducible nitric oxide synthase (iNOS) has been implicated in cancer formation because of its vast presence cancer tissues. Studies to support such a role during transformation of human cells are very limited. We have developed a cell culture system, which renders a more transformed epithelial phenotype. The model cells generated from immortalized human gingival mucosal (GM) keratinocytes are consisted of less transformed epithelial-like (EPI) cells and more transformed fibroblast-like (FIB) cells. The latter exhibit anchorage independent growth (AIG). Our data showed that iNOS at mRNA and protein levels was up-regulated in more transformed FIB cells in comparison with less transformed EPI cells. FIB cells at low passages (p<22) were unstable being able to morphologically and functionally revert back to EPI phenotype, while no reversion was observed in FIB cells at high passages (p>43). The morphological reversion of FIB cells was associated with the reversal of vimentin expression as well as AIG. More importantly, these revertants showed reduced levels of iNOS mRNA as well as MAP kinase ERK and phospho-ERK protein expression, while FIB cells without reversion maintained the expression. Furthermore, the MEK1/2 inhibitor U0126 could reduce detectable iNOS mRNA levels suggesting that MAP kinases were upstream regulators of iNOS transcription. U0126 caused both morphological and functional reversion of FIB cells indicating involvement of MAP kinases in these functions. Taken together, we provide evidence for an up-regulation of iNOS in cultured human keratinocytes which exhibit AIG. This up-regulation may reflect progressive transformation which still requires further changes to reach tumorigenic conversion.