Extracellular superoxide dismutase induces mouse embryonic fibroblast proliferative burst, growth arrest, immortalization, and consequent in vivo tumorigenesis.

AIMS: Rat sarcoma virus (RAS)-induced tumorigenesis has been suggested to follow a three-stage model consisting of an initial RAS activation, senescence induction, and evasion of p53-dependent senescence checkpoints. While reactive oxygen species act as second messengers in RAS-induced senescence, they are also involved in oncogenic transformation by inducing proliferation and promoting mutations. In the current work, we investigated the role of extracellular superoxide dismutase (SOD3) in RAS-induced senescence and immortalization in vitro and in vivo. We used a mouse embryonic fibroblast (MEF) primary cell model along with immortalized and transformed human cell lines derived from papillary and anaplastic thyroid cancer. RESULTS: Based on our data, sod3 RNA interference in H-RasV12-transduced cells markedly inhibited cell growth, while sod3 over-expression in MEFs initially caused a proliferative burst followed by the activation of DNA damage checkpoints, induction of p53-p21 signal transduction, and senescence. Subsequently, sod3-transduced MEF cells developed co-operative p21-p16 down-regulation and acquired transformed cell characteristics such as increased telomerase activity, loss of contact inhibition, growth in low-nutrient conditions, and in vivo tumorigenesis. Interestingly, as previously reported with RAS, we showed a dose-dependent response to SOD3 in vitro and in vivo involving transcriptional and non-transcriptional regulatory mechanisms. INNOVATION: SOD3 may mediate H-RasV12-induced initiation of primary cell immortalization. CONCLUSIONS: Our results indicate that SOD3 influences growth signaling in primary and cancer cells downstream of the ras oncogene and could serve as a therapy target at an early tumorigenesis phase.

Brief report: Mesenchymal stromal cell atrophy in coculture increases aggressiveness of transformed cells.

Mesenchymal stromal cells (MSCs) are able to influence the growth abilities of transformed cells. Here, we show that papillary thyroid cancer TPC1 and HEK 293T cells interact physically with human primary bone marrow-derived MSCs followed by evanescence of MSC cytoplasm. Interestingly, transformed cells were able to connect only to apoptotic MSCs that had lost their migration ability, whereas naïve MSCs avoided the direct contact. The interaction stimulated the proliferation of the cocultured transformed cells, activated mitogen and stress signaling, and increased resistance to cytotoxins. Consistent with in vitro data, the MSC interaction stimulated transformed cells had enhanced ability to grow and metastasize in vivo. The parental control cells showed mild tumorigenicity as compared to MSC interaction stimulated cells yielding measurable tumors in 31 days and 7 days, respectively. Our coculture model system describes how adjacent transformed cells absorb stromal cells thereby leading to the stroma-driven evolution of moderately carcinogenic cells to highly aggressive metastatic cells.

SOD3 decreases ischemic injury derived apoptosis through phosphorylation of Erk1/2, Akt, and FoxO3a.

BACKGROUND: Extracellular superoxide dismutase (SOD3), which dismutates superoxide anion to hydrogen peroxide, has been shown to reduce the free radical stress derived apoptosis in tissue injuries. Since both superoxide anion and hydrogen peroxide have a marked impact on signal transduction pathways and could potentially explain a number of apoptosis and survival -related phenomena in different pathological conditions, we clarified the impact of SOD3 on Akt and Erk1/2 cell survival pathways in rat hind limb injury model. METHODOLOGY AND PRINCIPAL FINDINGS: Based on our data, the hind limb ischemic rats treated with virally delivered sod3 have milder injury and less apoptosis than control animals that could be due to parallel activation of pro-proliferative and anti-apoptotic Erk1/2 and Akt pathways. The common downstream factor of both signaling pathways, the apoptosis related forkhead box protein O3a (FoxO3a), was phosphorylated and translocated to the cytoplasm in sod3 treated tissues and cell line. Additionally, we obtained increased mRNA production of elk-1, ets-1, and microRNA 21 (miR-21), whereas synthesis of bim mRNA was decreased in sod3 overexpressing tissues. We further showed that overexpression of sod3 modulated redox related gene expression by downregulating nox2 and inos when compared to injured control animals. CONCLUSIONS AND SIGNIFICANCE: The study shows the complexity of SOD3-derived effects on tissue injury recovery that are not limited to the reduction of superoxide anion caused cellular stress but highlights the impact of SOD3 related signal transduction on tissue functions and suggests an important role for SOD3 in attenuating cell stress effects in different pathological conditions.

Protective effects of hypothalamic proline-rich peptide and cobra venom Naja Naja Oxiana on dynamics of vestibular compensation following unilateral labyrinthectomy.

We tested the action of proline-rich peptide (PRP-1) and cobra venom Naja Naja Oxiana (NOX) on Deiters' nucleus neurons at 3rd, 15th and 35th days after unilateral labyrinthectomy (UL). Early and late tetanic, post-tetanic potentiation and depression of Deiters'neurons to bilateral high frequency stimulation of hypothalamic supraoptic and paraventricualar nuclei was studied. The analysis of spike activity was carried out by mean of on-line selection and special program. The complex averaged peri-event time and frequency histograms shows the increase of inhibitory and excitatory reactions of Deiters' neurons at early stage of vestibular compensation following PRP-1 and NOX injection, reaching the norm at the end of tests. In histochemical study the changes in Ca(2+)-dependent acidic phosphatase (AP) activity in neurons was discovered. It was shown that in UL animals the total disappearance or delay of decolorizing of Deiters' neurons lead to neurodegenerative pattern as cellular "shade". AP activity after UL and PRP-1 injection exerts more effective recovery of neurons in comparison with events, observed after the administration of NOX. The data of this study indicate that PRP-1 and NOX are protectors, which may successfully recover the disturbed vestibular functions.

Extracellular superoxide dismutase is a thyroid differentiation marker down-regulated in cancer.

Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), have a significant role in hormone production in thyroid tissue. Although recent studies have demonstrated that dual oxidases are responsible for the H(2)O(2) synthesis needed in thyroid hormone production, our data suggest a pivotal role for superoxide dismutase 3 (SOD3) as a major H(2)O(2)-producing enzyme. According to our results, Sod3 is highly expressed in normal thyroid, and becomes even more abundant in rat goiter models. We showed TSH-stimulated expression of Sod3 via phospholipase C-Ca(2+) and cAMP-protein kinase A, a pathway that might be disrupted in thyroid cancer. In line with this finding, we demonstrated an oncogene-dependent decrease in Sod3 mRNA expression synthesis in thyroid cancer cell models that corresponded to a similar decrease in clinical patient samples, suggesting that SOD3 could be used as a differentiation marker in thyroid cancer. Finally, the functional analysis in thyroid models indicated a moderate role for SOD3 in regulating normal thyroid cell proliferation being in line with our previous observations.

SOD3 reduces inflammatory cell migration by regulating adhesion molecule and cytokine expression.

Inflammatory cell migration characteristic of ischemic damages has a dual role providing the tissue with factors needed for tissue injury recovery simultaneously causing deleterious development depending on the quality and the quantity of infiltrated cells. Extracellular superoxide dismutase (SOD3) has been shown to have an anti-inflammatory role in ischemic injuries where it increases the recovery process by activating mitogen signal transduction and increasing cell proliferation. However, SOD3 derived effects on inflammatory cytokine and adhesion molecule expression, which would explain reduced inflammation in vascular lesions, has not been properly characterized. In the present work the effect of SOD3 on the inflammatory cell extravasation was studied in vivo in rat hind limb ischemia and mouse peritonitis models by identifying the migrated cells and analyzing SOD3-derived response on inflammatory cytokine and adhesion molecule expression. SOD3 overexpression significantly reduced TNFalpha, IL1alpha, IL6, MIP2, and MCP-1 cytokine and VCAM, ICAM, P-selectin, and E-selectin adhesion molecule expressions in injured tissues. Consequently the mononuclear cell, especially CD68+ monocyte and CD3+ T cell infiltration were significantly decreased whereas granulocyte migration was less affected. According to our data SOD3 has a selective anti-inflammatory role in ischemic damages preventing the migration of reactive oxygen producing monocyte/macrophages, which in excessive amounts could potentially further intensify the tissue injuries therefore suggesting potential for SOD3 in treatment of inflammatory disorders.

Extracellular superoxide dismutase is a growth regulatory mediator of tissue injury recovery.

Extracellular superoxide dismutase (SOD3) gene therapy has been shown to attenuate tissue damages and to improve the recovery of the tissue injuries, but the cellular events delivering the therapeutic response of the enzyme are not well defined. In the current work, we overexpressed SOD3 in rat hindlimb ischemia model to study the signal transduction and injury healing following the sod3 gene transfer. The data suggest a novel sod3 gene transfer-derived signal transduction cascade through Ras-Mek-Erk mitogenic pathway leading to activation of AP1 and CRE transcription factors, increased vascular endothelial growth factor (VEGF)-A and cyclin D1 expression, increased cell proliferation, and consequently improved metabolic functionality of the injured tissue. Increased cell proliferation could explain the improved metabolic performance and the healing of the tissue damages after the sod3 gene transfer. The present data is a novel description of the molecular mechanism of SOD3-mediated recovery of tissue injury and suggests a new physiological role for SOD3 as a Ras regulatory molecule in signal transduction.

The prevalence of the arcuate artery: a cadaveric study of 72 feet.

The purpose of this study was to quantify the occurrence of the arcuate artery. The arcuate artery was defined as that artery branching off the dorsalis pedis artery at or below the level of the tarsometatarsal joint, tending laterally across the bases of metatarsals 2 through 4, and supplying dorsal metatarsal arteries 2 through 4. The arcuate artery was present in 16.7% of 72 cadaver feet that were dissected and examined, suggesting that the arcuate artery is not the primary blood supply to dorsal metatarsal arteries 2 through 4 as is commonly described. It was determined that the lateral tarsal artery supplied dorsal metatarsal arteries 2 through 4 more frequently (47.2%) than the arcuate artery. The proximal perforating arteries as well as various combinations of all three sources were also found to contribute complete blood supply to dorsal metatarsal arteries 2 through 4. Therefore, a consistent dorsal arterial network, which differentiates throughout development, better explains the blood supply of the dorsal forefoot than the arcuate artery.

Epidemiology of HIV in Estonia.

The aim of this study was to report on the epidemiology of HIV infection in Estonia and to molecularly characterize Estonian HIV-1 variants. Epidemiological data were obtained from the Estonian National HIV/AIDS Database. In 1995-1996 blood samples were collected from 54 of the 65 HIV-infected individuals that had been diagnosed at that time. The V3 domain of the env gene was directly sequenced from peripheral blood mononuclear cells of 49 of these 54 individuals and the sequences used for phylogenetic analyses. At the end of 1999 Estonia reported 96 diagnosed HIV cases; 46 (48%) were homosexual or bisexual men and 31 (32%) were presumed to have been infected heterosexually. Importantly, only four individuals were likely to have become infected through intravenous drug use. Forty-three individuals (45%) were presumed to have been infected outside of Estonia, whereas 38 (40%) were likely to have become infected in Estonia. As expected, a majority of the investigated individuals (80%) were found to carry subtype B virus. Infections with subtypes A, C, D, G, and CRF02_AG were also documented. The dominance of subtype B was restricted to homosexual and bisexual men. Thus, subtype B infections were documented in 33 of 34 (97%) homosexual and bisexual men, but only 6 of 15 (40%) individuals with other probable routes of infection. Thirty of the 39 subtype B sequences were joined in a tight sequence cluster that also included sequences from neighboring Russia and Lithuania. This pattern suggests a local spread of HIV-1 among homosexual and bisexual men within the region. The results from the phylogenetic analyses agreed well with other epidemiological information. In conclusion, Estonia remains a country with a low prevalence of HIV infection. A majority of the identified cases are homosexual or bisexual men, whereas HIV infection among intravenous drug users is rare. A large proportion of the homosexual and bisexual men carried closely related subtype B variants. The sequences have been deposited in GenBank under accession numbers AF286538-AF286586.

An HIV type 1 subtype A outbreak among injecting drug users in Latvia.

Local, explosive outbreaks of HIV-1 infections among intravenous drug users (IDUs) caused by closely related variants have been reported in the Ukraine and Russia since 1996. Latvia experienced the start of a similar outbreak at the end of 1997 and already at the end of 1998 IDUs constituted approximately 50% of all known HIV cases (122 of 251). To investigate the molecular epidemiology of HIV-1 variants circulating among Latvian IDUs the V3 domain of the env gene was directly sequenced from samples of seven recently infected IDUs. Phylogenetic tree analysis showed that the seven Latvian HIV-1 variants had closely related subtype A genotypes that shared recent ancestry with each other as well as with variants involved in outbreaks in the Ukraine and southern Russia. There exists a clear risk for continued spread of HIV-1 in Latvia because the number of IDUs is increasing and needle sharing is common.

Assay of plasma samples representing different HIV-1 genetic subtypes: an evaluation of new versions of the amplicor HIV-1 monitor assay.

Quantification of plasma HIV-1 RNA levels has rapidly become the main tool for monitoring disease progression and treatment. However, some first-generation assays do not accurately quantify all HIV-1 subtypes. This study compares the first-generation and two newer prototype Amplicor HIV-1 Monitor assays (Roche Diagnostic Systems) in terms of their performance in quantifying HIV-1 RNA in stored plasma samples from 101 individuals infected with various genetic subtypes of HIV-1 (28 subtype A, 18 B, 26 C, 20 D, 2 E, 3 G, 2 H, and 2 J). The HIV-1 subtype had previously been determined by direct sequencing of the V3 domain of the env gene. The results from the three assays agreed for subtypes B and C, as well as for most subtype D samples. In contrast, the first-generation assay reported significantly lower plasma HIV-1 RNA levels than did the two newer versions of the assay for most subtype A, E, G, and H samples. There were no differences in mean plasma HIV-1 RNA levels between individuals infected with subtypes A, B, C, and D if the results from the two newer test versions were used, and if an adjustment was made between subtypes for differences in CD4 count. Thus, this study confirms that the first-generation assay does not accurately quantify HIV-1 RNA levels in many individuals infected with subtype A, E, G, and H. These problems appeared to have been greatly reduced in the two new prototype versions.

Reevaluation of the relaxed calcaneal stance position. Reliability and normal values in children and adults.

Reliability and normal values for the relaxed calcaneal stance position were determined in a nonclinic population of healthy adults and children (88 adults and 124 children) ranging in age from 5 to 36 years. The mean relaxed calcaneal stance position for adults was 6.07 degrees valgus (SD 2.71 degrees) (range, 1 degree varus to 14 degrees valgus). The mean relaxed calcaneal stance position for children was 5.6 degrees valgus (SD 2.9 degrees) (range, 6 degrees varus to 12 degrees valgus). There was no significant difference between the relaxed calcaneal stance positions of adults and children. In children the relaxed calcaneal stance position did not correlate with age, height, or weight and did not decrease with age to the theoretical normal value of 0 degree +/- 2 degrees as postulated by Root et al. The relaxed calcaneal stance position was found to be a reliable measurement; however, the theoretical normal value of 0 degree +/- 2 degrees was not found. The values reported in the present study correspond with the results of other empirical studies; thus the theoretical normal value for the relaxed calcaneal stance position of 0 degree +/- 2 degrees may be invalid.

Infectious causes of heel pain.

Heel pain is often attributed to a biomechanical etiology or sports-related injury. However, failure to recognize an infectious cause can lead to a delay in proper treatment and result in severe patient disability. This article reviews some of the more common infectious etiologies of heel pain.

Effect of a high dose of fluoride on resorbing osteoclasts in vivo.

Histomorphometric analysis of osteoclasts resorbing bone above the erupting first rat molar tooth germ was made at 1, 2, 6, 24, and 48 h following systemic administration of a single high dose of sodium fluoride. Compared to the number of osteoclasts in control rats, a significant reduction was observed in the number of actively resorbing osteoclasts in fluoride-injected rats at 2 and 6 h after dose administration, after which a gradual increase to control levels was observed. A locally increased number of inactive osteoclasts, not attached to bone surfaces, accompanied the reduced number of active osteoclasts.

Scanning electron microscopic study of orthodontically induced injuries to the periodontal membrane.

Scanning electron microscopy was used to study vascular and cellular changes in the periodontal membrane (PDM) in rats after orthodontic treatment ranging from 3 h to 9 days. The orthodontic forces ranged between 200 mN and 400 mN. Within the first hours of treatment extravasated erythrocytes, eichinocytes and platelets in various stages of degeneration were found in the pressure zones. The connective tissue of the pressure zones developed an amorphous appearance lacking discernible fibers and blood vessels following treatment for 24 h. These areas are known as hyaline zones, an area of local aseptic necorsis. Three days after the application of an orthodontic force macrophage-like cells were abundant close to the hyaline zone. They adhered to the hyaline zone with filopods radiating from pseudopods. Large osteoclast-like cells with several projections towards the bone surface were found around the hyaline zone and in the marrow spaces close to the pressure zones. The results indicated that the tissue reactions leading to remodeling of the PDM in the pressure zones are closely related to vascular injuries and subsequent formation of coagulated blood. Furthermore, macrophages seem to be the cells which play the most important role during degradation of the hyaline zone.

Alkaline phosphatase activity and tetracycline incorporation during initial orthodontic tooth movement in rats.

The activity of alkaline phosphatase and the incorporation of tetracycline as signs of bone formation were studied after orthodontic tooth movement for 10 h to 6 days in rats. Defined low or high forces were used. A moderate activity of non-specific alkaline phosphatase was found in the periodontal membrane (PDM) in untreated rats and in rats treated with low forces. In addition, all bone surfaces were outlined with a narrow band of intense non-specific alkaline phosphatase activity that was vanadate- and levamisole-resistant. Likewise, tetracycline was incorporated on all bone surfaces. The bone formation rate was low and uniform within the alveolus, indicating that no intra-alveolar drift of the molar occurred in the untreated rats. Orthodontic forces gradually inhibited vanadate- and levamisole-resistant alkaline phosphatases and tetracycline incorporation on the bone surfaces in the pressure zones of the PDM, depending on the magnitude of the force. It was suggested that the disappearance of these isoenzymes, in a limited area, as seen in the pressure zones, was associated with inhibited bone formation and subsequent initiation of bone resorption. On the tension side a slight reduction and redistribution of vanadate- and levamisole-resistant alkaline phosphatase activity could be noted irrespective of the magnitude of the applied force.

Cellular enzyme activity associated with tissue degradation following orthodontic tooth movement in man.

Enzyme histochemical techniques were used as markers of macrophage activity and differentiation in the periodontal tissues following orthodontic tooth movement in man. The enzymes studied included lactate dehydrogenase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, acid phosphatase and its tartrate resistant isoenzyme, arylsulfatase, aminopeptidase M and prostaglandin synthetase. Chloroacetyl esterase activity was studied in order to detect possible neutrophilic degrading activity. Intense activities of arylsulfatase and prostaglandin synthetase and a moderate activity of aminopeptidase M were found in cells degrading the hyaline zone. However, no activity of tartrate resistant acid phosphatase was found in these cells. Giant cells in contact with bone surfaces adjacent to the hyaline zone exhibited an intense activity of succinic dehydrogenase, tartrate resistant acid phosphatase and aminopeptidase M. Chloroacetyl esterase activity did not change following orthodontic treatment. The results indicate that macrophages in various stages of differentiation were responsible for the degradation of the hyaline zone and alveolar bone during orthodontic tooth movement. The enzymatic differences were probably due to the influence of the immediate cellular environment. Prostaglandin synthetase activity, which may be interpreted as a sign of prostaglandin secretion, was associated with the degradation of the hyaline zone in man.

An epidemiologic study of malignant oral tumors in Sweden 1958-77.

The paper presents epidemiologic data of malignant oral tumors in Sweden 1958-77 including the total number of cases, the number and relative frequency of cases in different sublocations of the oral region and the mean annual age standardized incidence rate, all ages, per one million population, by site and sex. An analysis of different histopathologic types of oral tumors is also presented. A comparison is made between the two 10-yr periods 1958-67 and 1968-77. Errors involved in epidemiologic studies are discussed.