Increased production of acute-phase proteins corresponds to the peak parasitaemia of primary malaria infection

Recent studies have implicated non-specific mediators associated with CD4⁺ T cells of the T helper 1 subset in resistance to experimental malarias. As part of continuing studies into the multifactorial role of nitric oxide and other contributors to the innate immune response in control of acute-phase malaria infection, the production of the acute-phase proteins, caeruloplasmin and serum amyloid P, following infection of naive mice with blood stages of the rodent malaria parasite Plasmodium chabaudi was investigated. Levels of both acute-phase proteins in the serum of infected mice were significantly elevated on days 7–12 post-infection compared both to other times of infection, and to background levels detected in uninfected control mice. These times corresponded to the ascending and peak primary parasitaemia, when production of interferon-γ, tumour necrosis factor- and nitric oxide is known to be raised. Although it is not apparent whether the production of caeruloplasmin and serum amyloid P has a causal effect in reducing parasitaemia or is simply a by-product of innate immunity, the detection of increased levels of circulating acute-phase proteins may act as a useful surrogate marker of high level parasitaemia, and therefore, of blood-borne malaria pathology.     

Expression of matrix metalloproteinase 9 in primary and recurrent breast carcinomas

The matrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases that are secreted as inactive precursors, which are activated by cleavage of an N-terminal pro-peptide. Their basic mechanisms of action include cancer cell growth, differentiation, apoptosis, migration and invasion, and the regulation of tumour angiogenesis and immune surveillance. The expression of MMP2 and MMP9 has been associated with high potential of metastasis in several human carcinomas including breast cancer. The 29 female patients, 9 premenopausal and 20 postmenopausal, aged from 37 to 79 years were included in this study. Tissue samples were examined in 29 primary and 48 recurrent carcinomas using the tissue microarrays which included 102 cores of primary breast carcinomas and 96 of recurrent breast carcinomas. Immunohistochemistry determined a pattern of expression for MMP9. The staining was diffuse cytoplasmic, strong, moderate, faint/weak and negative. The majority of the breast carcinomas stained homogenously for MMP9 on tumor cells. Statistically significant correlation was found for the expression of MMP9 between primary and recurrent breast carcinomas in general (p < 0.001) and in tumors that were grouped as recurrence before (p = 0.039) and after 24 months (p < 0.001). Strong expression of MMP9 was observed in primary tumors that recurred after 24 months, median: 162.5 (score range 0-300) and those tumors that recurred before 24 months of the initial diagnosis, median: 102.5 (score range 0-250) (p = 0.026).     

Glycolipid intermediates involved in the transfer of N-acetylglucosamine to endogenous proteins in a yeast membrane preparation.

A particulate membrane fraction from Saccharomyces cerevisiae contains transferases which catalyze the incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into a lipid fraction as well as into a protein fraction. The lipid fraction contains two alkali-stable lipids which can be separated on a silica G-60 column. The sugar moieties of these polyprenoid lipids are: N-acetylglucosamine and di-N-acetylchitobiose. The transfer of carbohydrate from isolated glycolipids to endogenous protein has been examined. After separation of protein and saccharide by hydrazinolysis and reacetylation only di-N-acetylchitobiose is found, and also when glycolipid containing only one N-acetylglucosamine is used as substrate. Maximum transfer of saccharides from glycolipids to protein is obtained at a Triton X-100 concentration of 1%. At this Triton X-100 concentration there is practically no transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the phosphorylated lipid. Therefore, when polyprenyl diphosphate N-acetyl[3H]-glucosamine is incubated together with UDP-N-acetyl[14C]glucosamine with the membrane fraction in the presence of 1% Triton X-100, a doubly labelled di-N-acetylchitobiose linked to lipid is formed with N-acetyl[14C]glucosamine at the non-reducing end of the chain.     

p53 Protein expression and proliferative activity in ductal breast carcinomas

The immunocytochemical expression of p53 protein and Ki-67 labelling index in tumour cells of 100 ductal breast carcinomas of different histological grade and stage was evaluated in cytological material. In order to investigate p53 expression and Ki-67 expression an avidin-extravidin immunocytochemical technique was applied to imprints. Monoclonal antibody (MoAb) DO-p53 and proliferating cell monoclonal antibody were used as primary antibodies. A statistically significant difference was observed between p53 protein expression and grade of malignancy and clinical stage (P = 0.001, P < 0.001, respectively). A statistically significant difference was also observed between Ki-67 LI and histological grade and stage of the tumours (P < 0.001, P < 0.001 correspondingly). A correlation was observed between p53 protein expression and Ki-67 LI (P < 0.001). The immunocytochemical study of p53 protein and Ki-67 expression in cytological material represents a simple method which can be applied in routine cytological laboratories for the investigation of potential malignancy of ductal breast cancer.     

Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas

Frequent chromosome 3 losses have been described in several tumors types, which strongly suggest the presence of one or several tumor suppressor genes. Recently, a novel candidate tumor suppressor gene termed Ris-1 (for Ras-induced senescence 1) has been identified at chromosomal position 3p21.3. Ris-1 has been proposed to participate in anti-tumor responses that resemble cellular senescence and that are elicited by oncogenes such as Ras. To analyze the role of Ris-1 as a putative tumor suppressor gene in human breast cancer, we have performed a real-time quantitative analysis of its mRNA expression in 60 patients. Moreover, we carried out a first approach to evaluate the most common inactivation mechanism that can affect expression levels of tumor suppressor genes (mutation, promoter hypermethylation and allelic losses). Furthermore, a correlation study between expression as well as inactivating mechanisms of Ris-1 and several clinico-pathological parameters of the tumors was designed, with the objective of appraising the prognostic value of Ris-1 status. Decreased expression of Ris-1 was observed in 23% of the cases and overexpressed Ris-1 was detected in 15% of the primary breast tumors. Our data showed high frequency of LOH (30%) at one of the markers used. Nevertheless, a polymorphism related with the expression levels was described. Statistically significant correlations were found between decreased Ris-1 expression and negative progesterone receptors, as well as between overexpressing Ris-1 tumors and high histological grade. Despite all these data, we conclude that the suggested role of Ris-1 as tumor suppressor gene is not evident, at least in breast cancer. Future and larger series studies in different tumor types are necessary to clarify Ris-1 function in human cancer.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

Increased transglycosylation activity of Rhodotorula glutinis endo-beta-glucanase in media containing organic solvent

The transglycosylation of p-nitrophenyl-beta-D-cellotrioside to cellotetraose catalyzed by endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture. Among various organic solvents tested, acetone was most effective. The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed. The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10 mM Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50 degrees C in the presence of 40% acetone.     

Variation of flow cytometric DNA measurement in 1,485 primary breast carcinomas according to guidelines for DNA histogram interpretation

From 1990-1996, 1,485 previously untreated invasive breast carcinomas were sampled by a pathologist for flow cytometric DNA analysis. The aim of the present work was to study the variations of flow cytometric DNA ploidy and S-phase evaluation according to the conditions of DNA histogram interpretation. Results obtained with the American Consensus guidelines of 1993 and the Fran?ois Baclesse Department of Pathology's own guidelines are presented. According to the percentage of events taken into account to identify a DNA aneuploid peak, the proportion of DNA diploid cases can change from 35-39%. For S-phase evaluation, although the two guidelines were quite different, the results of S-phase cutoff were identical. Whichever guidelines were used, there was a strong relationship between DNA ploidy and/or S-phase and classical clinicopathological factors (T, N, histological type, grade, receptor status, or lymphatic invasion), with the exception of age, whose correlation was discrepant with S phase according to the set of guidelines. Whichever guidelines were used, ploidy and S phase correlated strongly with survival (overall, metastasis-free, or recurrence-free). Hence we recommend the use of the American consensus guidelines, despite minor imperfections, because they are now well-known, allow a high yield in the ratio of assessable S phases, and permit standardization in the technical processing and reporting of S phases, thanks to the use of terciles.     

Allelic imbalance in primary breast carcinomas and metastatic tumors of the axillary lymph nodes

Axillary lymph node status is the most important prognostic factor in predicting disease outcome in women with breast cancer. A number of chromosomal aberrations in primary breast tumors have been correlated with lymph node status and clinical outcome, but chromosomal changes particular to metastatic lymph node tumors have not been well studied. DNA samples isolated from laser-microdissected primary breast and metastatic axillary lymph node tumors from 25 women with invasive breast cancer were amplified using 52 microsatellite markers defining 26 chromosomal regions commonly deleted in breast cancer. Levels and patterns of allelic imbalance (AI) within and between breast and lymph node tumors were assessed to identify chromosomal alterations unique to primary or metastatic tumors and to examine the timing of metastatic potential. The overall frequency of AI in primary breast tumors (0.24) was significantly greater (P < 0.001) than that in lymph node tumors (0.10), and congruent AI events were observed for < 20% of informative markers. AI at chromosomes 11q23.3 and 17p13.3 occurred significantly more frequently (P < 0.05) in primary breast tumors alone; no chromosomal regions showed a significantly higher AI frequency in lymph nodes. Higher rates of AI in primary versus metastatic lymph node tumors suggest that acquisition of metastatic potential may be an early event in carcinogenesis, occurring before significant levels of AI accumulate in the primary tumor. In addition, patterns of AI were highly discordant between tumor types, suggesting that additional genetic alterations accumulated independently in the two cell populations.     

Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (gamma-glutamylcysteine ligase heavy chain (gamma-GCL h), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and gamma-GCL h protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-beta-estradiol or ICI-182,780 did not significantly change gamma-GCL h expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE.     

Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.     

Increased blood spermine levels decrease the cytotoxic activity of lymphokine-activated killer cells: a novel mechanism of cancer evasion

Increased blood polyamine levels, often observed in cancer patients, have negative impacts on patient prognosis and are associated with tumor progression. The purpose of our study was to examine the effects of polyamines on cellular immune function. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured with the human natural polyamines spermine, spermidine, or putrescine, and the effects on immune cell function were examined. The correlation between post-operative changes in blood polyamine levels and lymphokine-activated killer (LAK) activity was also examined in cancer patients. Spermine decreased the adhesion of non-stimulated PBMCs to tissue culture plastic in a dose- and a time-dependent manner without affecting cell viability or activity. This decrease in adhesion capacity was accompanied by a decrease in the number of CD11a bright-positive and CD56 bright-positive cells. Upon stimulation with interleukin 2 to activate LAK cytotoxicity, PBMCs cultured overnight with 100 or 500 μM spermine showed decreased cytotoxic activity against Daudi cells (91.5 ± 1.7 and 84.9 ± 3.0%, respectively (n = 6) compared to PBMC cultured without polyamines). In a group of 25 cancer patients, changes in blood spermine levels after surgery were negatively correlated with changes in LAK cytotoxicity after surgery (r = −0.510, P = 0.008: n = 25). Increased blood spermine levels may be an important factor in the suppression of anti-tumor immune cell function.     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

乳腺、甲状腺多原发癌患者乳腺癌组织中RETmRNA的表达及意义

目的：研究乳腺癌术后发生甲状腺癌患者乳腺癌组织中RET（rearranged during transfection）基因的表达及临床意义。方法：用逆转录-聚合酶链反应（RT-PCR）检测33例乳腺、甲状腺多原发癌患者乳腺癌组织中RET基因的酪氨酸激酶区（TK）的表达进行分析,并设30例单纯乳腺癌和20例乳腺良性病变做对照组。结果：（1）乳腺、甲状腺多原发癌患者乳腺癌组织中RET-TK阳性检测率达57.6%（19/33）,单纯乳腺癌患者癌组织中RET-TK阳性检出率30%（9/30）,乳腺良性病变患者病变乳腺组织中RET-TK阳性检出率为10%（2/20）。三组的阳性率差别分别都有统计学意义（P＆lt;0.05）。（2）RET-TK的阳性表达与患者的ER情况相关（P＆lt;0.05）,而与患者年龄、肿瘤直径、同侧腋窝淋巴结转移、Her-2表达等病理学特征无关（P＆gt;0.05）。结论：RET基因的过量表达可能与乳腺、甲状腺多原发癌的发生有关。