Natural radioactivity of groundwater in Vendian deposits in St. Petersburg Region.

The study is focused on the hydrogeological conditions and the chemistry of groundwater of the Vendian aquifer in the western part of the Leningrad oblast (Karelian Isthmus and the area near Sosnovy Bor town) and St. Petersburg City, where groundwater features higher radioactivity, but nevertheless it is used for drinking water supply. Data on the radiological characteristics, which have been determined in the estimation of the quality of groundwater used for drinking are generalized and analyzed. These characteristics include the gross alpha and gross beta activity and the specific activity of natural radionuclides 222Rn, 226Ra, 228Ra, 210Pb, 210Po, 238U, and 234U. The data were subjected to statistical and correlation analysis to determine the hygienic criteria for the use of groundwater of this aquifer for drinking water supply and to study the sources and the processes of formation of the natural radiological background. Groundwater quality standards were shown to be exceeded in the majority of the analyzed wells. The brackish water in the southern, deeper, part of the aquifer system was shown to have higher radioactivity and relatively high concentrations of 226Ra, 228Ra, 210Pb, and 210Po, compared with fresh water in the northern part of the territory, of which higher, though nonuniform, 222Rn activity is typical. Relationships between the radiation characteristics of groundwater are considered along with the causes of formation of groundwater radionuclide composition as a result of the higher radioactivity of the host deposits and the chemistry of groundwater; changes in the radiological and hydrochemical background groundwater characteristics from the north to the south are characterized in accordance with the subsidence of the aquifer system and an increase in the stagnation of the hydrochemical regime. The analysis of the well-known relationship between the concentrations of radium isotopes in groundwater, uranium and thorium isotopes in the host rocks, and groundwater residence time in the aquifer, along with the comparison of the available field data with calculation results, suggested the conclusion that the concentration of uranium in the water-bearing rocks in the major portion of the area under consideration is higher than its regional mean values.

Hypoxia-inducible factor-1alpha expression requires PI 3-kinase activity and correlates with Akt1 phosphorylation in invasive breast carcinomas.

Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 and the key factor in cellular response to low oxygen tension. Expression of HIF-1alpha protein is associated with poor patient survival and therapy resistance in many types of solid tumors. Insight into HIF-1alpha regulation in solid tumors is important for therapeutic strategies. In this study, we determined the pathophysiological relevance of HIF-1alpha regulation by the oncogenic phosphatidylinositol 3'-kinase (PI 3-kinase)/Akt signaling pathway. We modeled the physiology of hypoxic tumor regions by culturing carcinoma cells under low oxygen tension in the absence of serum. We observed that hypoxic induction of HIF-1alpha protein was decreased by serum deprivation. Overexpression of dominant-active Akt1 restored HIF-1alpha expression, whereas inhibition of PI 3-kinase activity reduced hypoxic HIF-1alpha protein levels to a similar extent as serum deprivation. Immunohistochemical analysis of 95 human breast cancers revealed that lack of Akt1 phosphorylation correlates with low HIF-1alpha levels. To our knowledge, this is the first reported comparison between HIF-1alpha expression and Akt phosphorylation in human carcinomas. We conclude that Akt activity is physiologically relevant for HIF-1alpha expression in breast cancer. This implies that HIF-1alpha function might be therapeutically targeted by inhibition of the PI 3-kinase/Akt pathway.

Distinct regulation of p53 and p73 activity by adenovirus E1A, E1B, and E4orf6 proteins.

Multiple adenovirus (Ad) early proteins have been shown to inhibit transcription activation by p53 and thereby to alter its normal biological functioning. Since these Ad proteins affect the activity of p53 via different mechanisms, we examined whether this inhibition is target gene specific. In addition, we analyzed whether the same Ad early proteins have a comparable effect on transcription activation by the recently identified p53 homologue p73. Our results show that the large E1B proteins very efficiently inhibited the activity of p53 on the Bax, p21(Waf1), cyclin G, and MDM2 reporter constructs but had no effect on the activation of the same reporter constructs by p73, with the exception of some inhibition of the Bax promoter by Ad12 E1B. The repressive effect of the E1A proteins on p53 activity is less than that seen with the large E1B proteins, but the E1A proteins inhibit the activity of both p53 and p73. We could not detect significant inhibition of p53 functions by E4orf6, but a clear repression of the transcription activation by p73 by this Ad early protein was observed. In addition, we found that stable expression of the Ad5 E1A and that of the E1B protein both caused increased p73 protein expression. The large E1B and the E4orf6 proteins together do not target the p73 protein for rapid degradation after adenoviral infection, as has previously been found for the p53 protein, probably because the large E1B protein does not interact with p73. Our results suggest that the p53 and p73 proteins are both inactivated after Ad infection and transformation but via distinct mechanisms.

Adenovirus E1A proteins inhibit activation of transcription by p53.

p53 stimulates the transcription of a number of genes, such as MDM2, Waf1, and GADD45. We and others have shown previously that this activity of p53 can be inhibited by adenovirus type 2 or 12 large E1B proteins. Here we show that the adenovirus E1A proteins also can repress the stimulation of transcription by p53, both in transient transfections and in stably transfected cell lines. The inhibition by E1A occurs without a significant effect on the DNA-binding capacity of p53. Furthermore, the activity of a fusion protein containing the N-terminal part of p53 linked to the GAL4 DNA-binding domain can be suppressed by E1A. This indicates that E1A affects the transcription activation domain of p53, although tryptic phosphopeptide mapping revealed that the level of phosphorylation of this domain does not change significantly in E1A-expressing cell lines. Gel filtration studies, however, showed p53 to be present in complexes of increased molecular weight as a result of E1A expression. Apparently, E1A can cause increased homo- or hetero-oligomerization of p53, which might result in the inactivation of the transcription activation domain of p53. Additionally, we found that transfectants stably expressing E1A have lost the ability to arrest in G1 after DNA damage, indicating that E1A can abolish the normal biological function of p53.

Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells.

Previously, we have shown that the chicken anemia virus-derived VP3 ("apoptin") protein induces apoptosis in chicken mononuclear cells. Here, we report that apoptin also induces apoptosis in human osteosarcoma cells, regardless of whether they expressed wild-type, mutant p53, or no p53 at all. Moreover, the nuclear location of apoptin appears to be important for its optimal induction of apoptosis. The fact that apoptin can induce p53-independent apoptosis in human tumor cells makes apoptin a potential candidate for treatment of frequently occurring types of cancer cells that do not contain functional p53.

Wilms' tumor 1-KTS isoforms induce p53-independent apoptosis that can be partially rescued by expression of the epidermal growth factor receptor or the insulin receptor.

The Wilms' tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family. As a result of alternative RNA splicing, the gene can be expressed as four polypeptides that differ in the presence or absence of a stretch of 17 amino acids just NH2 terminal of the four zinc fingers and a stretch of three amino acids (+/-KTS) between zinc fingers 3 and 4. In this study, cDNA constructs encoding the four human Wilms' tumor 1 splice variants were transiently transfected into the p53-negative Hep3B and the p53-positive HepG2 hepatoma cell lines. Morphological assessment of the WT1-expressing cells showed that the WT1(-KTS) splice variants induced apoptosis in both cell lines, whereas the WT1(+KTS) isoforms did not. The induction of apoptosis by the WT1(-KTS) isoforms appears to be p53 independent in the hepatoma cell lines. Furthermore, it was found that the WT1(-KTS)-induced apoptosis could not be suppressed by coexpression of either the Mr 21,000 E1B, the Bcl-2, or the BAG-1 protein. Coexpression of either the epidermal growth factor receptor or the insulin receptor, however, partially rescued the cells from apoptosis.

Altered phosphorylation and oligomerization of p53 in adenovirus type 12-transformed cells.

Loss of function of the tumor-suppressor protein p53 is, in general, either caused by mutation, inducing a conformational change, or by binding to inactivating cellular (e.g. MDM2) or viral (e.g. SV40 large T) proteins. In adenovirus type 12 (Ad12)-transformed cells, p53 is stabilized without detectable binding to the Ad12 E1B/54 kDa protein and still present in a wild-type conformation but contains a mutant-like activity in cellular transformation. In this study we examined whether the changed characteristics of p53 in Ad12-transformed cells are correlated with changes in phosphorylation or complex formation of the protein. By making tryptic phosphopeptide maps we found a significant increase in the phosphorylation of the N-terminus of p53. Furthermore, expression of E1A was found to be essential for the altered phosphorylation, while expression of only Ad12 E1B/54 kDa is sufficient to increase the protein half-life. Additionally, we observed p53 to be present in increased molecular weight complexes in Ad12-transformed cells. We conclude that both the phosphorylation and oligomerization of p53 is changed as a result of Ad12 transformation.

Differential prognostic impact of hypoxia induced and diffuse HIF-1alpha expression in invasive breast cancer.

BACKGROUND: Intratumorous hypoxia triggers a broad cellular response mediated by the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1alpha concentrations increase during breast carcinogenesis, and are associated with poor prognosis. An earlier study noted two HIF-1alpha overexpression patterns: diffuse scattered throughout the tissue and confined to perinecrotic cells. AIMS: To investigate the prognostic impact of these different HIF-1alpha overexpression patterns in relation to its downstream effectors carbonic anhydrase (CA) IX and glucose transporter 1 (GLUT-1). METHODS: HIF-1alpha, CA IX, and GLUT-1 expression was studied by immunohistochemistry, including double staining for CA IX and HIF-1alpha. Clinical data included disease free survival, lymph node status, and tumour size. RESULTS: HIF-1alpha overexpression (44% of cases) had a perinecrotic (13.5%) or diffuse staining pattern (30.5%). CA IX expression was detectable in 12.5% of breast cancers, whereas GLUT-1 expression was seen in 29%, with both showing perinecrotic membrane staining. Perinecrotic HIF-1alpha overexpression was highly associated with CA IX and GLUT-1 overexpression, and double staining for HIF-1alpha and CA IX showed strong expression in the same cells. Diffusely overexpressed HIF-1alpha was not associated with CA IX or GLUT-1 expression. Patients with diffuse HIF-1alpha staining had a significantly better prognosis than patients with perinecrotically overexpressed HIF-1alpha. CONCLUSIONS: Different regulation pathways of HIF-1alpha overexpression exist in breast cancer: (1) hypoxia induced, perinecrotic HIF-1alpha overexpression with strong expression of hypoxia associated genes (CA IX and GLUT-1), which is associated with a poor prognosis; and (2) diffuse HIF-1alpha overexpression lacking major hypoxia associated downstream effects, resulting in a more favourable prognosis.

Hypoxia-induced acidification causes mitoxantrone resistance not mediated by drug transporters in human breast cancer cells.

Hypoxia has clinically been associated with resistance to chemotherapy. The aim of this study was to investigate whether hypoxia induces resistance to doxorubicin and mitoxantrone, two common drugs in cancer treatment, in MCF-7 breast cancer cells, and SW1573 non-small lung cancer cells. In addition, the role of drug transporters P-gp, BCRP and MRP1 was analysed. Hypoxia induced resistance in MCF-7 cells to mitoxantrone shifted the IC(50) value from 0.09 microM (+/-0.01) to 0.54 microM (+/-0.06) under hypoxia, whereas survival of MCF-7 and SW1573 cells in the presence of doxorubicin was not altered. Accumulation of mitoxantrone and daunorubicin, a doxorubicin fluorescent homologue, appeared to be 5.3 and 3.2 times lower in MCF-7 cells, respectively. Cytotoxicity assays showed no increased functionality of the drug transporters P-gp, BCRP and MRP1 under hypoxia. In addition, protein levels of these drug transporters were not changed. Medium of the MCF-7 cells became more acidic under hypoxia thereby causing a decreased uptake of mitoxantrone. Hypoxia induces mitoxantrone resistance in MCF-7 cells not mediated by the three major MDR transporters. Hypoxia-induced acidification may cause this resistance by decreased cellular uptake together with a lowered cytotoxicity due to pH-dependent topoisomerase type II activity.

Lack of lymphangiogenesis during breast carcinogenesis.

BACKGROUND: Recent evidence suggests that functional intratumorous lymph vessels may be absent from some human cancers. This could result from either the failure of tumours to induce lymphangiogenesis, or the collapse of lymph vessels, caused by high interstitial tumour pressure. METHODS: To differentiate between these two hypotheses, paraffin wax embedded clinical specimens from normal breast (n = 13), usual ductal hyperplasia (n = 11), ductal carcinoma in situ (n = 21), and invasive breast cancer (n = 40) were compared for lymphatic and blood vessel density by immunohistochemistry with antibodies to the lymphatic endothelial hyaluronan receptor (LYVE-1) and CD31, respectively. RESULTS: Lymph vessel density was lower than blood vessel density in normal breast tissue. Within breast lobuli, lymph vessels were absent. In premalignant lesions blood microvessel density increased, whereas no increase in lymph vessels could be seen intralesionally. In invasive cancers, lymph vessels were absent in all but a few cases, where probably some pre-existing lymph vessels remained, although blood microvessel density was once again increased. CONCLUSION: Unlike angiogenesis, lymphangiogenesis is absent during breast carcinogenesis. This, and not rising interstitial pressure caused by an increase in the size of lesions, explains the absence of intratumorous lymph vessels in invasive breast cancer.

[The administration of interleukin-1beta during early postnatal develop ment impairs FGF2, but not TIMP1, mRNA expression in brain structures of adult rats].

According to the Neurodevelopmental hypothesis, the long-lasting cognitive deficit in schizophrenia and other types of neuropathology may occur by injurious factors, such as hypoxia, traumas, infections that take place during pre- and postnatal development, at least at early stages. These pathological conditions are often associated with the high production of pro-inflammatory cytokine interleukin-1B (IL-1B) by the cells of immune and nervous systems. We investigated the expression of genes involved in the neuroplastic regulation (Fgf2 and Timp2) in medial prefrontal cortex and dorsal and ventral regions of hippocampus of adult rats that were treated with IL-1beta between P15 and P21. The learning impairment in IL-1beta-treated rats is accompanied by lower FGF-2 mRNA levels in medial prefrontal cortex and ventral (not dorsal) hippocampus, but TIMP-1 was not affected. No differences in TIMP-1 and FGF-2 mRNA expressions were observed in untrained IL-1beta-treated when compared to control rats.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha.

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFalpha protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFalpha) and aha-1 (HIFbeta), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2alpha-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2alpha, but not HIF-1alpha. These results identify TWIST1 as a direct target gene of HIF-2alpha, which may provide insight into the acquired metastatic capacity of hypoxic tumors.