Bortezomib downregulates MGMT expression in T98G glioblastoma cells.

The efficacy of treatment for glioblastoma multiforme is currently limited by the development of resistance, particularly, but not exclusively, due to the expression of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) in a significant proportion of astrocytic tumors. MGMT is post-translationally regulated by the 26S proteasome, a multi-subunit organelle responsible for degradation of misfolded cellular proteins. The boronic acid dipeptide bortezomib is the first and only proteasome inhibitor in clinical use so far, and has been reported as a strategy to restrict growth and promote apoptosis of glioblastoma cells. In this study we investigated the effect of bortezomib on MGMT expression in T98G cells, looking for an effect on the nuclear factor kappa B (NFκB) pathway, which is a major player in MGMT regulation and is also under tight control by the ubiquitin-proteasome system. Administration of bortezomib led to a significant reduction of T98G cell viability and induction of DNA fragmentation. These effects coincided with reduced expression of MGMT transcript levels, and a decrease in cellular amount and IκBα-mediated, proteasomal activity-dependent nuclear translocation of NFκB. In addition, bortezomib-induced phosphorylation of the translation initiation factor 2alpha (eIF2α) was in parallel with translational repression of MGMT. Taken together, these results suggest a novel role for bortezomib as a potent MGMT inhibitor and support its ongoing testing as a chemosensitizer in glioblastoma.

Additive inhibition of colorectal cancer cell lines by aspirin and bortezomib.

PURPOSE: To investigate the effect of cyclooxygenase-2 (Cox-2) inhibitor aspirin (acetylsalicylic acid, ASA) and proteasome inhibitor bortezomib in the proliferation and apoptosis of colorectal cancer cell lines. METHODS: MTT assay, trypan blue exclusion and DNA fragmentation have been used to investigate cell proliferation and apoptosis in the presence of drugs. For the determination of Cox activity a colorimetric method was used. Western blotting was used for the measurement of the effect of the drugs in different proteins expression. RESULTS: Bortezomib together with aspirin inhibit the growth of colorectal cancer cell lines HCT116, HT-29, and CaCo2 more than each drug alone. In the first two cell lines ASA inhibitory effects are Cox-2 independent because HCT116 cells do not express the enzyme while in HT-29 cells, Cox-2 has no activity as shown by a Cox activity assay. In CaCo2 cells that express enzymatically active Cox-2 this activity is inhibited by ASA. ASA is also able to suppress the increase in Cox-2 activity induced by bortezomib in these cells. Cell cycle inhibitors p21 and p27 are induced in the three cell lines by bortezomib and the combination treatment. Akt1 kinase is down-regulated in all three lines by the same treatments. Transcription factor NF-kappaB is retained in the cytoplasm by drug treatment in cell lines HCT116 and HT-29, a fact that may play a role in their pro-apoptotic activity. Pro-apoptotic bcl-2 family member, bad is down-regulated in cell lines HCT116 and CaCo2 by bortezomib treatment, a neoplasia-promoting event that is reversed by combination treatment. CONCLUSION: The combination of bortezomib and ASA cooperates to decrease proliferation and induce apoptosis in three human colorectal cell lines with different genetic lesions. These effects are at least in some cases Cox-2 independent and involve common and diverse mechanisms in the three lines.

Legionella species colonization of water distribution systems, pools and air conditioning systems in cruise ships and ferries.

BACKGROUND: Legionnaires' disease continues to be a public health concern in passenger ships. This study was scheduled in order to investigate Legionella spp. colonization of water distribution systems (WDS), recreational pools, and air-conditioning systems on board ferries and cruise ships in an attempt to identify risk factors for Legionella spp. colonization associated with ship water systems and water characteristics. METHODS: Water systems of 21 ferries and 10 cruise ships including WDS, air conditioning systems and pools were investigated for the presence of Legionella spp. RESULTS: The 133 samples collected from the 10 cruise ships WDS, air conditioning systems and pools were negative for Legionella spp. Of the 21 ferries WDS examined, 14 (66.7%) were legionellae-positive. A total of 276 samples were collected from WDS and air conditioning systems. Legionella spp. was isolated from 37.8% of the hot water samples and 17.5% of the cold water samples. Of the total 96 positive isolates, 87 (90.6%) were L. pneumophila. Legionella spp. colonization was positively associated with ship age. The temperature of the hot water samples was negatively associated with colonization of L. pneumophila serogroup (sg) 1 and that of L. pneumophila sg 2 to 14. Increases in pH >/=7.8 and total plate count > or =400 CFU/L, correlated positively with the counts of L. pneumophila sg 2 to 14 and Legionella spp. respectively. Free chlorine of > or =0.2 mg/L inhibited colonization of Legionella spp. CONCLUSION: WDS of ferries can be heavily colonized by Legionella spp. and may present a risk of Legionnaires' disease for passengers and crew members. Guidelines and advising of Legionnaires' disease prevention regarding ferries are needed, in particular for operators and crew members.

Hemopoietic progenitor cells and bone marrow stromal cells in patients with autoimmune hepatitis type 1 and primary biliary cirrhosis.

BACKGROUND/AIMS: Autologous hematopoietic stem cell transplantation has been used in severe cases of autoimmunity. We investigated whether hemopoietic progenitor cells and/or bone marrow (BM) microenvironment are affected in autoimmune hepatitis type-1 (AIH-1) and primary biliary cirrhosis (PBC). METHODS: We studied 13 AIH-1 patients, 13 PBC patients, 12 cirrhotic controls (CC) and ten healthy controls (HC). Flow cytometry, expansion cultures, long-term BM cultures and clonogenic progenitor cell assays were used. Stromal cell function was assessed in long-term BM cultures recharged with normal CD34+ cells. RESULTS: AIH-1 had increased CD34+, CD34+/CD38+ and CD34+/CD38- cells compared to all groups (P<0.001). PBC had lower progenitor cells compared to controls (P<0.005). No differences were found between CC and HC. Committed progenitor cells in non-adherent cell fraction were increased in AIH-1 (P<0.05) but decreased in PBC compared to controls (P<0.05). Granulocyte-macrophage colony forming units (CFU) and erythroid-burst CFU were increased in AIH-1 compared to all groups (P<0.001). PBC had these CFUs decreased compared to controls (P<0.005). Stromal cells failed to support normal hemopoiesis in PBC. CONCLUSIONS: We demonstrated for the first time that AIH-1 had increased hemopoietic progenitor cells and normal stromal function. In PBC, progenitor cells and BM microenvironment were defective. Further studies will determine the significance of these novel findings.

Diabetes mellitus and lung function.

OBJECTIVES: To assess the nature of pulmonary dysfunction in type 1 diabetes and the relationship of pulmonary function tests to diabetic factors and complication. SUBJECTS AND METHODS: Sixteen type 1 diabetic patients and 26 control subjects matched for age and sex were studied. We performed spirometry measurements and measured pulmonary diffusing capacity (DL(CO)) in sitting and supine position by the single-breath method corrected by alveolar volume (VA). Glycosylated hemoglobin (HbA(Ic)), retinopathy and microalbuminuria were included as parameters of metabolic control and diabetic complications. RESULTS: Diabetic patients showed a significant reduction of the following pulmonary function tests (% predicted value) as compared with control subjects: total lung capacity (TLC, 92.6 +/- 14.5 vs. 113.9 +/- 17.5, p < 0.001), lung diffusing capacity in sitting position (DL(CO), 90.4 +/- 21.1 vs. 107.7 +/- 15.6, p = 0.004), lung diffusing capacity in supine position (DL(CO), 88.3 +/- 19.3 vs. 111.9 +/- 19.9, p = 0.001). The differences in diffusing capacity corrected by alveolar volume in sitting and supine position (DL(CO)/VA) were not significant. By changing the posture from sitting to supine position both diabetic patients and control subjects significantly increased DL(CO)/VA (103.4 +/- 17.7 vs. 112.7 +/- 22.3, p = 0.046 and 99.5 +/- 13.4 vs. 114.4 +/- 13, p < 0.001, respectively). There was no correlation between pulmonary function tests and diabetic complications. CONCLUSION: These data indicate that type 1 diabetic patients showed reduced TLC and DL(CO), features of pulmonary restrictive dysfunction. There was no correlation between abnormal pulmonary function and the presence of other diabetic complications.