A Study of the Cytotoxic Effect of Microcapsules and Their Constituent Polymers on Macrophages and Tumor Cells.

We studied the effect of different concentrations of polyelectrolytes poly(allylamine hydrochloride) (PAH) and polystyrene sulfonate (PSS) as well as the effects of microcapsules coated with these polymers on survival of Ehrlich ascites carcinoma cells and mouse peritoneal macrophages and on ROS production by phagocytes. PAH reduced viability of Ehrlich ascites carcinoma in a concentration-dependent manner (LD50=12-15 µg/ml). This effect was presumably determined by its ability to bind phosphates, thereby depleting the culture medium. At the same time, PAH did not affect the viability of macrophages. PSS produced no cytotoxic effect on the examined cells. Polyelectrolyte capsules with the shell architectonics (PAH/PSS)3 and (PAH/PSS)3PAH in the examined concentration range had no effect on the viability of macrophages and tumor cells. PAH microcapsules with positively charged surface much more rapidly and more intensively activated macrophages. The chemiluminescence response directly depended on the amount of capsules in the solution.

[Effect Of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase].

Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 M and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect.

Ultraviolet B(UVB)-induced cox-2 expression in murine skin: an immunohistochemical study.

Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostaglandins from arachidonic acid. This enzyme exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles. However, COX-2 expression is induced by a variety of agents, which include pro-inflammatory agents and mitogens. Evidence exists to indicate that increased expression of COX-2 occurs in several types of epithelial neoplasms. In this study, we show the effect of chronic exposure of murine skin to carcinogenic UVB on cutaneous COX-2 expression. SKH-1 mice were irradiated with 180 mJ/cm(2) UVB daily for five days a week for periods ranging from 1 to 20 weeks. Nontumor bearing skin areas of irradiated mice, skin of age-matched controls and benign papillomas and malignant tumors were assessed immunohistochemically for COX-2 expression in these mice. No epidermal staining occurred in any of the non-UVB-treated controls throughout the experiment. Epidermal COX-2 expression only occurred in UVB-irradiated mice. After 1 and 5 weeks of irradiation, patchy epidermal staining mostly confined to the granular layer and stratum corneum was observed. At week 9, staining intensity had increased, particularly in the granular layer. At week 13, staining was uniformly seen in all epidermal layers with particular prominence in the basal cell layer underlying areas of visible epidermal hyperplasia. It is of interest that the most intense staining was seen in the perinuclear region of keratinocytes and at the plasma membrane. At week 20, COX-2 staining was predominant in the granular layer, although in some tissue sections, the entire epidermis was positive. In benign papillomas, staining was confined to the superficial layers of the epidermis and in squamous cell carcinomas (SCCs), patchy staining in the granular and spinous layers predominated. In general, COX-2 expression was more intense in well-differentiated SCCs than in papillomas. In summary, our results indicate that COX-2 serves as an early marker of epidermal UVB exposure and its expression increases in benign papillomas and in SCCs. These results suggest that pharmacological intervention using specific COX-2 inhibitors could have anticarcinogenic effects in UVB-induced human skin cancer.

Concurrent acute lymphoblastic leukemia and juvenile pilocytic astrocytoma in a pediatric patient.

The concurrence of acute lymphoblastic leukemia (ALL) and an asymptomatic juvenile pilocytic astrocytoma is described. A 6-year-old boy without clinical evidence of neurofibromatosis had a juvenile pilocytic astrocytoma diagnosed on radiologic examination and before treatment of acute pre-B cell lymphoblastic leukemia. The patient has had a partial resection of the astrocytoma and is 9 months into treatment of his ALL, which is in complete remission. p53 gene mutation was not identified in this patient. The concurrent diagnosis before treatment of ALL and juvenile pilocytic astrocytoma, the latter normally an indolent tumor, suggests that some cases of astrocytoma previously ascribed to radiotherapy or other treatment may in fact be caused by other factors.

Mechanism of ultraviolet B-induced cell cycle arrest in G2/M phase in immortalized skin keratinocytes with defective p53.

The ultraviolet B (UVB) portion (280-320 nm) of solar radiation is considered to be a major etiologic factor in human skin cancer and is a known cause of extensive DNA damage. In this study, we observed that UVB exposure of immortalized epidermal keratinocytes (HaCat cells) harboring mutant p53 leads to G(2)/M cell cycle arrest in both asynchronously growing and synchronized cells in a dose dependent manner. Following UVB exposure (200 mJ/cm(2)), we observed a threefold increase in G(2)/M population at 6 h, which increased to sixfold. The observed G(2)/M arrest was associated with an increase in cyclin B level whereas cdc2 protein remained unchanged. However, we observed an accumulation of tyrosine 15 hyperphosphorylated cyclin B-cdc2 complex. In addition, we observed an increase in chk1 kinase and a decrease in cdc25C protein levels. Chk1 phosphorylates cdc25C on serine 216 and inactivates it whereas cdc25C dephosphorylates tyrosine 15 phosphate of cdc2 and activates the cdc2-cyclin B complex. Therefore, the increase in chk1 and the decrease in cdc25C both participate in inhibiting the G2/M transition. Our data identifies two upstream targets leading to inhibition of cyclin B-cdc2 complexes, which explain the inhibition in cyclin B-associated cdc2 kinase following UVB exposure. The inactive phosphorylated cdc2-cyclin B complex remains sequestered in cytoplasm and may migrate to the nucleus following activation. Our data also indicate that UVB exerts unique effects in different types of skin keratinocytes having nonfunctional or mutant p53.

Inequalities in health. Analytic approaches based on life expectancy and suitable for small area comparisons.

STUDY OBJECTIVE: Simple measures of inequalities in health are proposed to facilitate the work of health policy makers and to build on the understanding of health differences between populations. In addition, it is aimed to make these measures applicable for comparisons of small populations and subgroups. METHODS: Inequalities in health or health deficiencies were quantified as the difference between the life expectancy of the subgroup of interest and that of the national population. Health deficiencies were divided into disease specific components by partial application of cause eliminated life table methods. To manage small numbers and to depict time trends, locally weighted regression smoothing was applied. Confidence intervals were constructed through Monte Carlo simulations. APPLICATIONS AND COMPARISONS: The proposed approaches were applied to the health situation in Cape Breton County, Nova Scotia, Canada, and disclosed the significance of different diseases and distinct patterns between communities. The proposed measures were also compared with the traditionally used standardised mortality rates and ratios. Here, the proposed measures appeared beneficial in that they are easier to comprehend and that they provide time trends and more robust estimates. CONCLUSIONS: The above advantages make the proposed approaches beneficial to health policy makers and epidemiologists. The approaches may also be incorporated in economic evaluations as well as in more sophisticated public health models.

Formation of nuclear Bax/p53 complexes is associated with chemotherapy induced apoptosis.

Mechanisms by which chemotherapeutic agents induce apoptosis are not completely understood. Current knowledge of the actual pharmacologic effects of chemotherapy and their biochemical mechanisms are better understood than the downstream events, which initiate the apoptotic cascade. The chemotherapeutic agent cisplatin causes DNA damage and can induce apoptosis in several types of human cancers. We found the formation of previously unreported nuclear complexes between the tumor suppressor protein p53 and the pro-apoptotic protein Bax, in human melanoma cell lines induced into apoptosis following cisplatin exposure. These detergent resistant complexes were detected: after wild type (wt) p53 and Bax increased in the nucleus; at the same time when active cytoplasmic apoptosis related protease, caspase 3/CPP32 appeared; and prior to the detection of apoptotic DNA fragmentation. Three channel fluorescence laser scanning confocal image microscopy revealed that the nuclear Bax/p53 complexes remained in the nucleus and localized proximal to DNA fragmentation sites as assayed by TUNEL after cisplatin exposure. Two human melanoma cell lines, expressing wt p53, were induced into apoptosis after cisplatin exposure, however they differed in the timing of this induction. In both cell lines the formation of nuclear Bax/p53 co-immunoprecipitable complexes correlated with the timing of the induction of apoptosis. The degree of apoptosis induced by different concentrations of cisplatin correlated with the amount of nuclear Bax/p53 complexes. The co-immunoprecipitation of Bax and p53 was found regardless of the antibodies tested and was specific since Bcl-xL/p53 complexes were not detected. Additionally, the human prostate cancer cell line, LNCaP, also formed nuclear Bax/p53 complexes only after apoptosis was induced by paclitaxel.

Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells.

A p53-derived C-terminal peptide induced rapid apoptosis in breast cancer cell lines carrying endogenous p53 mutations or overexpressed wild-type (wt) p53 but was not toxic to nonmalignant human cell lines containing wt p53. Apoptosis occurred through a Fas/APO-1 signaling pathway involving increased extracellular levels of Fas/FasL in the absence of protein synthesis, as well as activation of a Fas/APO-1-specific protease, FLICE. The peptide activity was p53-dependent, and it had no effect in three tumor cell lines with null p53. Furthermore, the C-terminal peptide bound to p53 protein in cell extracts. Thus, p53-dependent, Fas/APO-1 mediated apoptosis can be induced in breast cancer cells with mutant p53 similar to the recently described Fas/APO-1 induced apoptosis by wt p53. However, mutant p53 without p53 peptide does not induce a Fas/APO-1 activation or apoptosis. Docking of the computed low energy conformations for the C-terminal peptide with those for a recently defined proline-rich regulatory region from the N-terminal domain of p53 suggests a unique low energy complex between the two peptide domains. The selective and rapid induction of apoptosis in cancer cells carrying p53 abnormalities may lead to a novel therapeutic modality.

An imperfect correlation between DNA replication activity of Epstein-Barr virus nuclear antigen 1 (EBNA1) and binding to the nuclear import receptor, Rch1/importin alpha.

Epstein-Barr virus (EBV) replicates as a stable multicopy episome in latently infected mammalian cells. Latent cycle DNA replication requires only two viral elements, the cis-acting origin of plasmid replication (oriP) and the trans-acting origin binding protein (EBNA1). EBNA1 binds multiple recognition sites in oriP, but has not other enzymatic activities associated with replication functions. To identify human cellular proteins that mediate EBNA1 function, we designed a one-hybrid assay in yeast to select for proteins that bind to EBNA1 when bound to criP in vivo. A human cDNA encoding the Rch1/hSRP1 alpha/ importin alpha protein was isolated and shown to bind to full-length EBNA1, but not to an amino terminal deletion mutant of EBNA1 when bound to oriP in yeast. The interaction of EBNA1 with Rch1 was confirmed biochemically by coimmunoprecipitation from nuclear extracts and by direct binding of recombinant proteins in vitro. Internal deletion mutations in EBNA1 which compromised DNA replication activity were similarly reduced for binding to Rch1. Mutations with no effect on DNA replication activity were similarly unaffected for Rch1 binding. Rch1/importin alpha has been shown to bind to the nuclear localization sequence (NLS) of several proteins and stimulate nuclear import. A substitution mutation in the EBNA1 nuclear localization sequence reduced Rch1 binding, but had no effect on DNA replication function, indicating that Rch1 binding affinity does not correspond precisely with replication activity. Nevertheless, the identification of a stable interaction between Rch1 and EBNA1 at the origin of viral DNA replication raises the intriguing possibility that Rch1 contributes to the nuclear functions of EBNA1.

Cultures of airway parasympathetic nerves express functional M2 muscarinic receptors.

To study the control of acetylcholine release from airway parasympathetic neurons, primary cultures of these cells were established. Guinea pig tracheas were disaggregated with collagenase and plated onto matrigel-coated plates in medium that contained cytosine arabinoside to inhibit growth of dividing cells. Over 7 to 10 days neurites grow from the cell bodies, reaching a length of 2 mm. The vast majority of the cells in these cultures were neurons, as identified by morphology and staining with Neurotag and with antibody to neuron-specific antigen protein gene product 9.5. Cultured neurons contained acetylcholine, which was released by electrical field stimulation. Thus these were parasympathetic neurons. Staining with antibodies to M1, M2, and M4 muscarinic receptors revealed the presence of only M2 receptors. Likewise, reverse transcription-polymerase chain reaction using primers for M1, M2, and M4 muscarinic receptors revealed mRNA only for M2 receptors. Blocking these M2 receptors using atropine potentiated the stimulated release of acetylcholine, demonstrating that the M2 receptors inhibit acetylcholine release, as they have been shown to do in vivo. Thus airway parasympathetic neurons can be grown in culture, they retain the ability to synthesize and release acetylcholine, and they express functional inhibitory M2 muscarinic receptors.