Biomedical subjects

Lorenza Bellincampi

Publications and source records attributed to Lorenza Bellincampi.

9 recordsLinked to original sources

PubMed2006

Role of GSTP1-1 in mediating the effect of As2O3 in the Acute Promyelocytic Leukemia cell line NB4.

Arsenic trioxide (As2O3) is a highly effective agent in the treatment of acute promyelocytic leukemia (APL), whereas other hematopoietic tumors are less responsive to this agent and mechanisms underlying As2O3,-resistance are poorly understood. To better understand the complex network of GSH-related pathways in As2O3 sensitivity, we investigated the role of GSH and GSH-relevant enzymes in an APL cell line sensitive to As2O3 (NB4) and in a resistant subclone (AsR). Cell proliferation, viability, and apoptosis were investigated in NB4 cells before and after treatment with 1 muM As2O3 and in AsR cells. In these experimental cell models, GSTP1-1, JNK1 and JNK2 proteins were analyzed by immunoblotting, and a kinase assay for JNK1 was performed. GSH levels as well as the activities of the enzymes glutathione peroxidase, glutathione transferase, gamma-Glutamylcysteynilsinthetase and superoxide dismutase were measured. NB4 cells treated with As2O3 showed a high level of oxidative stress and an increase of GSH levels. GSTP1-1 polymerization and JNK1 activation were detectable after 24 h and were followed by an increase of the apoptotic rate starting at 72 h. Neither GSTP1-1 polymerization nor JNK activation was found in AsR cells that showed a very low apoptotic rate. Our results suggest that APL sensitivity to As2O3 might be, at least in part, mediated by the balance between association and dissociation of JNK from GSTP1-1, depending on the redox status of the cell. Further investigation is warranted to find a way to interfere with this balance, whenever it might represent a mechanism of drug resistance.

Antineoplastic Agents↗

PubMed2005

Role of gamma-glutamyl cysteine synthetase (gamma-GCS) gene expression as marker of drug sensitivity in acute myeloid leukemias.

BACKGROUND: Elevated levels of glutathione (GSH) have been reported to play an important role in mediating chemoresistance in tumor cells. The regulation of gamma-glutamylcysteine synthetase (gamma-GCS) is one of the major determinants of GSH homeostasis. The aim of our study was to investigate gamma-GCS gene expression in patients affected by acute myeloid leukemia (AML). METHODS: A total of 64 AML samples, including 23 acute promyelocytic leukemia (APL or M3) cases, were included in the study. gamma-GCS mRNA levels were determined by real-time quantitative RT-PCR. All patients were evaluated at diagnosis, whereas post-treatment gamma-GCS mRNA levels were assessed at the end of the consolidation therapy in 16 cases. RESULTS: Our data showed that variable degrees of gamma-GCS expression were detectable in AML, likely reflecting disease heterogeneity; in particular, APL cases, compared to the other AML subsets, showed both significantly lower basal levels of gamma-GCS mRNA at presentation and significantly increased mRNA levels after treatment. CONCLUSIONS: Decreased levels of gamma-GCS leading to reduced GSH may at least in part explain the higher sensitivity of APL to chemotherapy.

Acute Disease↗

PubMed2005

Glutathione S-transferase P1 *C allelic variant increases susceptibility for late-onset Alzheimer disease: association study and relationship with apolipoprotein E epsilon4 allele.

BACKGROUND: Oxidative stress and neuronal cell death have been implicated in the pathogenesis of Alzheimer disease (AD). Considering that the glutathione transferase (GST) supergene family encodes isoenzymes that appear to be critical in protection against oxidative stress, we aimed at determining the various GSTP1, GSTM1, and GSTT1 polymorphisms and ApoE genotypes to investigate their role as susceptibility genes for late-onset AD (LOAD). METHODS: We included 210 LOAD patients and 228 healthy controls matched for age, sex, and educational level in our case-control genetic association study. GSTM1 and GSTT1 genotypes were studied by conventional PCR, whereas GSTP1 and ApoE genotypes were determined by real-time PCR on the LightCycler. RESULTS: We found a significant association between LOAD and the GSTP1*C allelic variant [odds ratio (OR) = 1.9; P < 0.05], but no association between the GSTM1 and GSTT1 deleted genotypes and LOAD. In addition, a preliminary result suggested that carriers of both the GSTP1*C and ApoE epsilon4 allelic variants were at increased risk of LOAD (OR = 19.98; P < 0.0001). CONCLUSION: The GSTP1*C allelic variant should be considered a candidate for LOAD, particularly in persons having the ApoE epsilon4 allelic variant, because the GSTP1 and ApoE gene products are implicated in oxidative stress and apoptosis processes leading to beta-amyloid-mediated neurodegeneration.

Age of Onset↗

PubMed2005

A proteomic investigation into etoposide chemo-resistance of neuroblastoma cell lines.

Neuroblastoma, one of the most common pediatric solid tumors, originates from the peripheral sympathetic nervous system and is responsible for approximately 15% of all childhood cancer deaths. Among the several antineoplastic drugs used in neuroblastoma chemotherapeutic protocols, topoisomerase inhibitors (i.e., etoposide) represent the most commonly used. Several resistance mechanisms limit the clinical success of topoisomerase-targeting drugs, mainly reducing the ability of neoplastic cells to start programmed cell death when exposed to antineoplastic drugs. The aim of this study was to determine, by means of proteomics, potential markers of etoposide resistance in human neuroblastoma cell lines as well as to investigate protein levels and modifications possibly involved in the onset of resistance. The etoposide resistant clone showed overexpression of the following proteins: peroxiredoxin 1, beta-galactoside soluble lectin binding protein, vimentin (three protein spots), heat shock 27 kDa protein (two protein spots) and heterogeneous nuclear ribonucleoprotein K. In addition, we also found down-regulation of dUTP pyrophosphatase. This investigation might represent a first step towards the development of novel prognostic markers of neuroblastoma chemotherapy.

Antineoplastic Agents, Phytogenic↗

PubMed2004

Hypermethylation of the CpG islands in the promoter region of the GSTP1 gene in prostate cancer: a useful diagnostic and prognostic marker?

BACKGROUND: Recently, many studies have focused on the potential diagnostic value of the promoter hypermethylation of the GSTP1 gene in prostate cancer. METHOD: A total of 144 patients, undergoing eight-core prostatic biopsies for a clinically suspected prostate cancer, was analyzed. Two different tissue samples were collected from the same area of the prostate and then divided for both genomic DNA extraction and pathological examination. In order to perform molecular analysis, prostatic tissue samples were digested with the methylation-sensitive restriction enzyme HpaII and then amplified by conventional polymerase chain reaction (PCR). RESULTS: Prostate cancer was diagnosed in 42/144 patients, and promoter hypermethylation of GSTP1 gene was detected in 31/42 of prostate cancer (sensitivity=74%) and in 2/102 of negative specimens (specificity=98%). A significant association between GSTP1 promoter hypermethylation both with a Gleason score >or=7 (Fisher's exact P=0.01) and the presence of Gleason grade 4 and/or grade 5 (Fisher's exact P=0.03) was found. CONCLUSION: Promoter hypermethylation of the GSTP1 gene is a highly specific--but not a very sensitive--marker of prostate cancer. Our data showed a significant association between the methylation status of the GSTP1 gene and Gleason score and grade, suggesting a potential prognostic value of this epigenetic DNA alteration.

Aged↗

PubMed2004

Screening of antinuclear antibodies: comparison between enzyme immunoassay based on nuclear homogenates, purified or recombinant antigens and immunofluorescence assay.

Current clinical practice considers antinuclear antibody (ANA) testing as a screening test; this has a major impact on laboratory work with a growing volume of analyses that need to be performed rapidly, to maintain good specificity and sensitivity. Ongoing discussions have been raised in order to identify the best technology to use in ANA screening, taking into account both clinical and economical implications. The aim of our study was to compare three different enzyme immunoassays (EIA) with immunofluorescence (IF) assay in order to identify which test is better for use as a screening test. The study was performed on 473 sera and the three different EIA tests were based on nuclear homogenates from HeLa cells, purified antigens from HEp-2 cells and recombinant antigens, respectively. The concordance between EIA-ANA and IF-ANA techniques, determined by the K statistic, was acceptable, but not complete, and discrepancies between both EIA-positive/IF-negative samples and IF-positive/EIA-negative were found. Both methods show interesting diagnostic abilities, however, the IF-ANA assay seems to be the first choice test in a well-standardized immunofluorescence laboratory with experienced microscopists, whereas the EIA test might be useful especially in large-scale ANA screening.

Animals↗

PubMed2003

Analysis of GSTP1-1 polymorphism using real-time polymerase chain reaction.

BACKGROUND: Glutathione transferases (GSTs) play an important role in the protection of cells from the products of oxidative stress as well as from several environmental carcinogens. The GSTP1-1 gene class is significantly overexpressed in many human tumors. Four allelic variants have been described for the GSTP1-1 gene (*A, *B, *C, *D) leading to different amino acid substitutions in position 105 and 114 of the protein sequence. The proteins encoded by the different alleles show different abilities to metabolize carcinogens and anticancer agents, suggesting an association between GSTP1 polymorphism and the risk for a variety of cancers as well as between said polymorphism and varying responses to cancer treatments. METHODS: The GSTP1-1 polymorphism was determined using a real-time polymerase chain reaction (PCR) and fluorescence resonance energy transfer with a Light-Cycler Instrument. ARMS was used in the case of *A/*C or *B/*D heterozygosity. We used this method to determine the GSTP1-1 polymorphism in 250 free-living Italian subjects of both sexes. RESULTS: Among 250 subjects representative of an Italian population, we observed the following allelic frequencies: f(A)=0.710, f(B)=0.236, f(C)=0.054 and f(D)=0. The observed phenotypes are in Hardy-Weinberg equilibrium (chi(2)=0.71, df=4, P=0.95). CONCLUSIONS: We have extended and improved a method of GSTP1-1 complete genotyping. This method provides the ability to genotype 30 samples in 2 h and it represents a fast, reliable and automated methodology to determine GSTP1-1 polymorphism in order to perform large-scale population studies.

Adult↗

PubMed2002

pRb2/p130 promotes radiation-induced cell death in the glioblastoma cell line HJC12 by p73 upregulation and Bcl-2 downregulation.

This study shows that in the glioblastoma hamster cell line HJC12 the retinoblastoma family member pRb2/p130 enhances gamma-radiation-induced cell death. In HJC12 cells the tetracycline-regulated expression of pRb2/p130 increased the percentage of gamma-radiation-induced apoptotic cells from 27 to 47%. pRb2/p130 overexpression was associated with the downregulation of the anti-apoptotic factor Bcl-2 and the upregulation of the steady-state protein levels of the pro-apoptotic transcription factor p73. In particular, RT-PCR showed a significant increase in the expression of the p73delta isoform when pRb2/p130 was overexpressed. The ability of pRb2/p130 to modulate apoptosis was not associated with its role in mediating G0/G1 arrest during cell cycle progression. Our data suggest a role for pRb2/p130 in glioblastoma gamma-radiation-induced cell death, indicating that the antitumoral action of pRb2/p130 can regulate both inhibition of cell cycle progression and induction of cell death.

Animals↗

PubMed2002

Apolipoprotein E genotyping: a comparative study between restriction endonuclease mapping and allelic discrimination with the LightCycler.

BACKGROUND: The apolipoprotein E (apo E) polymorphism is associated with the risk of developing cardiovascular disease and the risk and the time of onset of Alzheimer's disease. Therefore, the interest in apo E genotyping is high, both for epidemiological research and for the purpose of diagnosing dyslipidemia or dementia. The aim of our study was to compare and evaluate two different methods for apo E genotyping, both on the basis of polymerase chain reaction (PCR). METHODS: Genomic DNA of 197 subjects was extracted from whole blood. The first method involved DNA amplification performing a PCR using specific primers and endonuclease restriction mapping. The second one was a DNA assay that used real-time PCR on the LightCycler instrument (Roche). RESULTS: We obtained a 100% concordance between the two methods and we found a relative allelic frequency distribution typical for an Italian population. CONCLUSIONS: The LightCycler (LC) allelic discrimination method for apo E genotyping seems to be rapid, simple and accurate, suggesting a possible successful use of this method for diagnostic purposes.

Alleles↗