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Helicobacter pylori infection leads to chronic gastritis and increased risk of gastric cancer. The mechanism involves chronic inflammation. We aimed to determine the mechanism by which H. pylori infection causes telomere shortening in inflammatory gastric mucosa. Gastric biopsy specimens were obtained from 20 patients with chronic gastritis or peptic ulcer caused by H. pylori infection. The specimens showed increased NF-κB and superoxide dismutase activities and elevated expressions of PARP-1 and γ-H2AX, all of which returned to normal levels after anti-H. pylori treatment, suggesting that oxidative DNA damage and PARP-1 overexpression might cause telomere shortening. In this report, we adopted DNA end joining assay and showed that H. pylori-infected gastric mucosa had increased alternative NHEJ (non-homologous end joining), implicating that telomere shortening was caused by inflammation-mediated overproduction of reactive oxygen species and PARP-1, leading to telomere shortening.

Human sorcin,Mr22-kDa/pI5.3, was identified in HOB1 lymphoma cells resistant to 1.0 μ M vincristine (designated HOB1/VCR1.0). The protein was purified and polyclonal antibody against it was produced. Using antibody to precipitate sorcin from32P-labeled cell extract, the SDS–PAGE-resolved human sorcin was not phosphorylated in contrast to the report of A. M. van der Blieket al.((1986)EMBO J.5, 3201–3208) in which the authors emphasized hamster sorcin might be phosphorylated through a cAMP-dependent protein kinase. Although sorcin had not been found among proteins of parental HOB1 cells labeled by [35S]methionine and resolved by 2-D PAGE, it could be immunoprecipitated from the cytosolic extract of the same cells.In vitrophosphorylation study did not reveal an enhanced phosphoprotein located in the area of 22 kDa, either. A 521-bp human sorcin cDNA fragment synthesized by PCR was used to screen cDNA library constructed from mRNA of HOB1/VCR1.0 cells. Human sorcin cDNA encoded 198 amino acids, 10 among which have been replaced in hamster sorcin. Of the 10 amino acid changes, 6 involve serine's in human sorcin compared to hamster's 1. These data suggest a difference in phosphorylation state between the two species. The results of Southern blot indicate the sorcin gene was amplified in HOB1/VCR1.0 cells under the pressure of high concentration of vincristine but not secondary to themdrgene amplification. 2-step PCR, exhibiting the gene could be latently expressed in parental HOB1 cells, confirms the above result of immunoprecipitation. Interestingly, HOB1/VCR0.5 cells (resistant to 0.5 μ M vincristine) do not show amplification of the gene but do have increased expression of the protein. When this cell line was cultured in 0.1 μ M vincristine for a period of time, the protein's expression amount reverted to the latent level. These results suggest that sorcin should have a place in mediating resistance of HOB1 cells to vincristine.

Hsp90 (Heat shock protein-90) is a cellular buffer against erroneous gene products and also plays an essential role in facilitating proper folding, maturation, and activity of its client proteins. The phosphatidylinositol-3 kinase (PI-3K)-Akt pathway transduces a survival signal involved in tumor development. The kinase activity of Akt depends on its association with Hsp90. Hsp90 inhibition causes Akt degradation, but the mechanism remains unclear. Several reports showed that the Hsp90 inhibitor geldanamycin (GA) induces Thr308 and Ser473 phosphorylations of Akt, however, it is still unknown about the significance of GA-induced Akt activation in degradation of the kinase. We treated Hela cells with GA to observe Akt degradation and found that LY294002 delayed Akt degradation. Mutation of Thr308 or Ser473 also caused delayed Akt ubiquitination and degradation. Inhibition of Akt dephosphorylation enhanced GA-mediated Akt degradation. In this report, we show that GA-mediated transient activation of Akt accelerates its association with the E3 ligase CHIP (C-terminal Hsp70-interacting protein)-mediated ubiquitination and subsequent proteasome degradation.

A multidrug-resistant lymphoma cell line resistant to 1.0 microM vincristine (designated HOB1/VCR1.0) was established. The tubulins of parental and resistant cell lines were purified by ion-exchange chromatography. Two-dimensional polyacrylamide gel electrophoresis of tubulins showed a decrease in the basic component of beta-tubulin in the HOB1/VCR1.0 cell line; native isoelectric focusing of tubulins showed decreased expression of two more basic tubulin dimers in the same cell line. The [3H]vincristine-tubulin binding studies were performed by filtration and HPLC and displayed the tubulin of HOB1/VCR1.0 cells having a weaker binding affinity to vincristine than those of parental HOB1 and HOB1/VCR0.5 cells. The binding constant Ka of purified tubulin to vincristine, calculated from the slope of the Scatchard curve, for parental HOB1 cells was 5.6 x 10(6), and that for HOB1/VCR1.0 cells was 3.1 x 10(6). The Scatchard kinetics was also used to determine the binding ability of the purified tubulins to [3H]colcemid: the Kas for parental and HOB1/VCR1.0 cells were 3.9 x 10(5) and 2.0 x 10(5), respectively. The current study suggests that high-level resistant cells, HOB1/VCR1.0, tend to express fewer tubulin isoforms of stronger binding affinities to antimitotic agents; that is, they preserve weak drug-binding forms rather than produce additional species. This may be a mechanism for the cells to protect themselves from drug injury when the P-glycoprotein cannot efficiently pump out the agent of high concentration within the cells.

The X protein of hepatitis B virus (HBx) has been specifically implicated in either pro-apoptotic or anti-apoptotic activity in an experimental system, but the underlying mechanism is yet uncertain. Activations of survival and proliferation signaling pathways appear to account partly for its anti-apoptotic property. Change in mitochondrial membrane potential may be responsible for its apoptotic property. In this study, we isolated two HBx isoforms from an HBV carrier, one of which contains Akt phosphorylation site at Ser31 and functions as an anti-apoptotic protein (designated HBx-S31). The other does not contain Akt phosphorylation site and functions as an apoptotic protein (designated HBx-L31). HBx-S31 can activate Akt, whereas HBx-L31 cannot; the former enhances tumor growth, whereas the latter suppresses tumorigenesis. Our study provides evidence that HBx plays dual roles, namely pro-apoptotic and anti-apoptotic, through different isoforms in which HBx with Ser31 transduces survival signal.