Chemokine gene expression in rat pancreatic acinar cells is an early event associated with acute pancreatitis

BACKGROUND & AIMS: The molecular mechanisms underlying pancreatitis are largely unknown. The goal of this study was to identify an early genetic event that correlated with pancreatitis. METHODS: Differential display of messenger RNAs (mRNAs) was conducted on normal pancreas vs. those of animals with secretagogue-induced pancreatitis. Northern blots from normal animals and animals with experimental acute pancreatitis were probed with cloned complementary DNAs for chemokines. Pancreatitis was induced with cerulein and by retrograde injection of bile salts. Immunocytochemistry was used to identify the source of chemokine expression. Pyrrolidine dithiocarbamate was tested for effects on chemokine expression and pancreatitis. RESULTS: A differentially amplified band was consistently observed early after cerulein hyperstimulation. This band was identified as a portion of the mob-1 gene, an alpha-chemokine. Northern analysis indicated that mRNAs for mob-1 and another chemokine, mcp-1, were induced after cerulein hyperstimulation in vivo. mob-1 mRNA was also induced by retrograde injection of bile salts and by cerulein in acinar cells in vitro. mob-1 protein was localized to exocrine cells in pancreata of diseased animals. Pyrrolidine dithiocarbamate inhibited both chemokine gene expression and early inflammatory characteristics of pancreatitis. CONCLUSIONS: Chemokines are induced in acinar cells by treatments that induce pancreatitis and may play an important role in the early stages of the disease.     

DNA topoisomerase II alpha expression is associated with alkylating agent resistance

Increased expression of DNA topoisomerase II alpha has been associated with resistance to certain DNA-damaging alkylating agents, but no causal relationship or mechanism has been established. To investigate this observation, we developed a model of topoisomerase II overexpression by transfecting a full-length Chinese hamster ovary topoisomerase II alpha into EMT6 mouse mammary carcinoma. Topoisomerase II alpha-transfected cell lines demonstrated continued topoisomerase II alpha mRNA and protein expression, which were undetectable in vector-only lines, in stationary phase (G0-G1). The topoisomerase II transfectants were approximately 5-10-fold resistant to the alkylating agents cisplatin and mechlorethamine. Upon release from G0-G1, the topoisomerase II transfectants demonstrated more rapid thymidine incorporation and shorter cell-doubling times than control cells. Purified topoisomerase II and nuclear extracts with topoisomerase II-decatenating activity bound to cisplatin-treated DNA with significantly greater affinity than to untreated DNA in a cisplatin concentration-dependent manner. These observations suggest that expression of topoisomerase II alpha may have a role in cellular resistance to antineoplastic alkylating agents. The mechanism for this may involve increased binding of topoisomerase II alpha to alkylating agent-damaged DNA.     

Increased expression of the NME1 gene is associated with metastasis in epithelial ovarian cancer

The genetic events involved in the development of metastases of epithelial ovarian cancer are largely unknown. One gene postulated to play a role in tumour metastasis suppression is NME1 (nm23-H1), and an inverse relationship between NME1 expression and metastatic potential has been observed for some solid tumours. In this study we have investigated the levels of mRNA expression of the 2 isoforms of the NME gene, NME1 and NME2. A maximum of 45 tumours samples from 33 patients were available for Northern blot analysis. We observed variable levels expression of NME1 and NME2 mRNA. The average level of NME1, but not NME2, mRNA expression was statistically higher in metastatic biopsies when compared with primary tumour biopsies. To examine the possible tumour suppressor gene role of NME1 in ovarian tumours, 76 patients were investigated by Southern blot analysis to determine the rate of allelic deletion. Allele loss at 5 other chromosome 17 loci (D17S5, TP53, NF1, D17S74, D17S4) was also evaluated for many of these 76 patients. Allele loss was observed in 22/30 (73%) informative patients at the NME1 locus. We also observed high rates of allele loss at the other loci evaluated. No correlations with clinical stage, histological subtype or patient survival were observed in either mRNA or DNA analyses. We have established that tumour progression in ovarian cancer is accompanied by over-expression of the NME1 gene; however, despite high rates of allele loss at the NME1 locus, the concept that NME1 may be a candidate tumour suppressor gene in ovarian cancer cannot be confirmed by this study.     

Increased level of p21 in human ovarian tumors is associated with increased expression of cdk2, cyclin A and PCNA

We have demonstrated over-expression of the cyclin-dependent kinase inhibitor p21 in various ovarian-cancer cell lines as well as in ovarian-tumor biopsies. This increase in p21 expression relative to that observed in normal ovarian epithelial cells is unrelated to proliferation index. In the present study, we found that p21 is functional, since the protein extracted from IGROVI cells is still able to inhibit cdk2-kinase activity. We then investigated how IGROVI cells overcome the growth-inhibitory function of p21. Immunofluorescence assays and subcellular fractionation showed that p21 is located in cytoplasm and nucleus both in normal and in tumoral cells. Compared with normal ovarian epithelial cells in culture, the increase in level of p21 in IGROVI cells was found to be associated with increased expression of cdk2, cyclin-A and PCNA proteins. In IGROVI cells, p21 is associated with inactive cdk2/cyclin-A complex, indicating that it acts as an inhibitory factor rather than an assembly factor. Over-expression of cdk2 and of cyclin A observed in IGROVI cells allows them to escape to p21-inhibitory activity. The fact that cells from ovarian-tumor biopsies exhibited a concomitant increase in p21 and in its partners cdk2 and PCNA suggest that ovarian-tumor cells can tolerate high levels of functional p21 via over-expression of other cell-cycle-regulatory proteins.     

Increased plasminogen binding is associated with metastatic breast cancer cells: differential expression of plasminogen binding proteins

Overexpression of urokinase-type plasminogen activator and its receptor correlates with metastatic capacity in breast cancer. In this study we show that the urokinase/urokinase receptor-overexpressing, metastatic human breast cancer cell line MDA-MB-231 (1) bound significantly more cell-surface plasminogen in a lysine-dependent manner and (2) was capable of generating large amounts of plasmin compared with the non-metastatic cell lines MCF-7 and T-47D. In addition, distinct plasminogen binding proteins were detected in the plasma membranes of the cell lines, suggesting heterogeneity of binding proteins. Plasminogen binding was analysed using a combination of dual-colour fluorescence flow cytometry and ligand histochemistry (for comparative and cellular localization of ligand binding), and fluorimetry (for Scatchard analysis). Apart from revealing the greater plasminogen binding capacity of MDA-MB-231 cells, flow cytometry and histochemistry also revealed that, in all three cell lines, non-viable or permeabilized cells bound significantly more plasminogen in a lysine-dependent manner than viable or non-permeabilized cells. Viable MDA-MB-231 cells bound plasminogen with moderate affinity and high capacity (Kd = 1.8 microM, receptor sites per cell 5.0 x 10(7). Our results indicate that differences in cell surface-specific plasminogen binding capacity between cell lines may not be detectable with binding techniques that cannot distinguish between viable and non-viable cells.     

Increased ratio of bcl-2/bax expression is associated with bovine leukemia virus-induced leukemogenesis in cattle

To further investigate the molecular basis underlying the dysregulation of B cell homeostasis associated with bovine leukemia virus disease progression in cattle, bovine bax was cDNA cloned and sequenced. The predicted amino acid sequence of bovine Bax revealed a 192-amino-acid protein having extensive identity with the human (97%), murine (93%), and rat (94%) homologues. Because the ratio of Bcl-2 to Bax is believed to predetermine the susceptibility to a given apoptotic stimulus, the relative expression of the genes encoding these oncoproteins was evaluated in cattle naturally infected with BLV. In BLV-infected cattle an increase in the ratios of bcl-2/bax mRNA and protein expression correlated with advancing stages of disease. These findings suggest that in addition to the maintenance of BLV-associated hematopoietic malignancies, the reciprocal expression of Bcl-2/Bax may modulate the induction of B cell expansion typical of BLV disease progression.     

Baculovirus-mediated expression and characterization of the full-length murine DNA methyltransferase

The original cDNA sequence reported for the murine DNA methyltransferase (MTase) was not full length. Recently, additional cDNA sequences have been reported that lie upstream of the original and contain an extended open reading frame with three additional ATGs in frame with the coding region [Tucker et al . (1996) Proc. Natl. Acad. Sci. USA , 93, 12920-12925; Yoder et al . (1996) J. Biol. Chem . 271, 31092-31097]. Genomic DNA upstream of this ATG contains two more ATGs in frame and no obvious splice site. We have constructed, and expressed in baculovirus, MTase clones that begin at each of these four ATGs and examined their properties. Constructs beginning with any of the first three ATGs as their initiator methionines give a predominant DNA MTase band of approximately 185 kDa on SDS-PAGE corresponding to translational initiation at the third ATG. The fourth ATG construct gives a much smaller protein band of 173 kDa. The 185 kDa protein was purified by HPLC, characterized by mass spectrometry and has a measured molecular mass of 184 +/- 0.5 kDa. All of these MTases were functional in vitro and steady state kinetic analysis showed that the recombinant proteins exhibit similar kinetic properties irrespective of their length. The homogeneous recombinant enzyme from the fourth ATG construct shows a 2.5-fold preference for a hemi-methylated DNA substrate as compared to an unmethylated substrate, whereas the 185 kDa protein is equally active on both substrates. The kinetic properties of the recombinant enzyme are similar to those reported for the native MTase derived from murine erythroleukemia cells. The new clones are capable of yielding large quantities of intact MTases for further structural and functional studies.     

Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation

The organoselenium compounds benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC), as well as sodium selenite, are effective chemopreventive agents for various chemically induced tumors in animal models at both the initiation and postinitiation stages. The mechanisms involved at the postinitiation stage are not clear. Because several lines of evidence indicate that inhibition of excess DNA (cytosine-5)-methyltransferase (Mtase) may be a sufficient factor for the suppression or reversion of carcinogenesis, we examined the effects of sodium selenite, BSC, p-XSC and benzyl thiocyanate (BTC), the sulfur analog of BSC, on Mtase activity in nuclear extracts of human colon carcinomas, and of p-XSC on the Mtase activity of HCT116 human colon carcinoma cells in culture. For this purpose, we developed an improved Mtase assay, in which the incorporation of the methyl-[3H] group from S-adenosyl[methyl-3H]methionine into deoxycytidine of poly(dI-dC)-poly(dI-dC), is specifically determined by HPLC with radioflow detection after enzymatic hydrolysis, enhancing specificity and reliability. In a variation, using SssI methyltransferase and labeled S-adenosylmethionine, the overall methylation status of DNA in various tissues can also be compared. Selenite, BSC and p-XSC inhibited Mtase extracted from a human colon carcinoma with IC50s of 3.8, 8.1 and 5.2 microM, respectively; BTC had no effect. p-XSC also inhibited the Mtase activity and growth of human colon carcinoma HCT116 cells, with an IC50 of approximately 20 microM. The improved Mtase assay should prove to be a reliable method for screening potential Mtase inhibitors, especially using cells in culture. We suggest that inhibition of Mtase may be a major mechanism of chemoprevention by selenium compounds at the postinitiation stage of carcinogenesis.     

Increased expression of heat-shock protein 47 is associated with overproduction of type I procollagen in systemic sclerosis skin fibroblasts

Albicidins are important factors in systemic pathogenesis by Xanthomonas albilineans, which causes the devastating leaf scald disease of sugar cane. They are also of substantial interest as antibiotics that selectively block prokaryote DNA replication. Albicidin biosynthesis is highly sensitive to medium composition. An optimized, chemically defined medium (SMG3) yielded 30-fold more albicidin from half the accumulated biomass, relative to sucrose peptone (SP) medium. Phosphate starvation stimulated albicidin production in SMG3 and SP media. Addition of other amino acids, ammonium ions or peptones to the defined medium increased the growth rate of X. albilineans XA3, but differentially inhibited albicidin biosynthesis. Knowledge of these factors indicates new approaches to understanding mechanisms of pathogenesis and resistance to sugar cane leaf scald disease, and to strain improvement for production of albicidin antibiotics.     

Creutzfeldt-Jakob disease associated with autonomic nervous system dysfunction in the early stage

A 54-year-old man presented with tremor and unusual behavior. He was admitted two months later because of dementia and myoclonus. Periodic synchronous discharges were observed on the electroencephalogram. Based on these findings, we diagnosed the case as Creutzfeldt-Jakob disease. About two weeks after admission, decubitus, bowel dysfunction and hypohidrosis occurred. We observed various autonomic nervous system dysfunctions such as abnormal pupillary response to autonomic drugs, reduced coefficient of variation of R-R interval, and abnormal diurnal blood pressure variation.     

Increased interhemispheric interaction is associated with earlier offset of childhood amnesia.

Experiment 1 found that mixed-handedness, which is associated with increased interaction between the left and right cerebral hemispheres relative to strong right-handedness, was associated with an earlier offset of childhood amnesia. In Experiment 2, bilateral saccadic eye movements, which have been shown to enhance interhemispheric interaction, were also associated with an earlier offset of childhood amnesia. These results build upon a growing body of research indicating an interhemispheric basis for the retrieval of episodic memories. Moreover, the results of Experiment 2 suggest that interhemispheric interaction has its effect on the retrieval, not encoding, of episodic memories. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Coexistent thyroiditis is associated with lower tumour stage in thyroid carcinoma

BACKGROUND: Microsatellite instability (MSI) is a new mechanism described in carcinogenesis of several tumours and seems to predispose for cancer in some chronic inflammatory diseases. As thyroiditis is thought to be both the cause and the consequence of thyroid carcinoma, it was the aim of this retrospective study to investigate the epidemiology and the influence of a coexistent thyroiditis on prognosis and clinicopathological parameters in an iodine-deficient area. METHODS: The grade of lymphocytic infiltration (LI) of 153 thyroid carcinomas was determined in paraffin-embedded tissues: G0 = no LI, G1 = peritumoral LI, G2 = peritumoral LI with follicle, G3 = diffuse LI. A non-radioactive PCR-based screening method was used for detection of MSI. RESULTS: Twenty-seven (17.7%) out of 153 carcinomas were accompanied by thyroiditis (G1, 16; G2, 5; G3, 6). Ten cases fulfilled the criteria necessary for diagnosing Hashimoto's thyroiditis. Nine out of 10 (90%) Hashimoto's cases and 16 out of 17 (94%) other thyroiditis cases were associated with a significantly (chi2 < 0.01) lower pT stage (pT1, pT2) than cases without thyroiditis. No statistical association was found by comparing multifocality or sclerosing variants with the grade of lymphocytic infiltration. MSI was detected neither in patients with severe inflammation nor in the absence of thyroiditis. CONCLUSIONS: MSI does not seem to play a role in the pathogenesis of thyroid carcinoma. Coexistent thyroiditis is associated with a lower pT stage and thus could be an indicator of a better prognosis.     

Modified oligonucleotides as bona fide antagonists of proteins interacting with DNA. Hairpin antagonists of the human DNA methyltransferase

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. Ex vivo, hairpin inhibitors of DNA MeTase are localized to the cell nucleus in lung cancer cells. They inhibit DNA MeTase, cell growth, and anchorage independent growth (an indicator of tumorigenesis in cell culture) in a dose-dependent manner. The inhibitors developed in this study are the first documented example of direct inhibitors of DNA MeTase in living cells and of modified oligonucleotides as bona fide antagonists of critical cellular proteins.     

Reduced expression of cyclin-dependent kinase inhibitor p27Kip1 is associated with advanced stage and invasiveness of gastric carcinomas

Reduced expression of a cyclin-dependent kinase inhibitor p27Kip1 has recently been shown to predict poor survival of patients with breast and colorectal cancers. We studied the expression of p27Kip1 in gastric carcinomas by northern blotting, western blotting and immunohistochemistry to determine whether lack of p27 has implications for aggressiveness of gastric cancer. Reduced expression of p27 was detected in 40% of the gastric carcinomas at the mRNA level, while it was detected in 57% at the protein level. No gross alterations of the p27 gene were observed in any of the cases examined by Southern blot analysis. Immunohistochemical studies revealed that the expression of p27 was well preserved in most of the gastric adenomas, whereas it was so in only 26% of the gastric carcinomas. Fifty-six percent of the carcinomas showed almost no p27-positive cells. Decrease of p27-positive cells significantly correlated with advanced stage, depth of tumor invasion and lymph node metastasis. The expression of p27 showed an inverse correlation with the expression of cyclin E. These findings suggest that reduction of p27Kip1 protein may reflect the progression of gastric carcinomas and may be an indicator of high-grade malignancy.     

Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas

Malignant astrocytomas, which are highly invasive, vascular neoplasms, compose the majority of nervous system tumors in humans. Elevated expression of fibroblast growth factors (FGFs) in astrocytomas has implicated the FGF family of mitogens in the initiation and progression of astrocyte-derived tumors. In this study, we demonstrated that human astrocytomas undergo parallel changes in FGF-receptor (FGFR) expression during their progression from a benign to a malignant phenotype. FGFR type 2 (BEK) expression was abundant in normal white matter and in all low-grade astrocytomas but was not seen in malignant astrocytomas. Conversely, FGFR type 1 (FLG) expression was absent or barely detectable in normal white matter but was significantly elevated in malignant astrocytomas. Malignant astrocytomas also expressed an alternatively spliced form of FGFR-1 (FGFR-1 beta) containing two immunoglobulin-like disulfide loops, whereas normal human adult and fetal brains expressed a receptor form (FGFR-1 alpha) containing three immunoglobulin-like disulfide loops. Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR-2 and a shift in expression from FGFR-1 alpha to FGFR-1 beta as they progressed from benign to malignant phenotype. These results suggest that differential expression and alternative splicing of FGFRs may be critical in the malignant progression of astrocytic tumors.