Nuclear localization of NADPH:cytochrome c (P450) reductase enhances the cytotoxicity of mitomycin C to Chinese hamster ovary cells

Overexpression of endoplasmic reticulum-localized NADPH: cytochrome c (P450) reductase (NPR) in Chinese hamster ovary cells increases the hypoxic/aerobic differential toxicity of the mitomycins. Because considerable evidence indicates that DNA cross-links are the major cytotoxic lesions generated by the mitomycins, we proposed that bioactivation of the mitomycins in the nucleus close to the DNA target would influence the cytotoxicity of these drugs. The simian virus 40 large T antigen nuclear localization signal was fused to the amino-terminal end of a human NPR protein that lacked its membrane anchor sequence. Immunofluorescent imaging of transfected cell lines expressing the fusion protein confirmed the nuclear location of the enzyme. Regardless of the oxygenation state of the cell, mitomycin C (MC) cytotoxicity was enhanced in cells with overexpressed NPR localized to the nuclear compartment compared with cells overexpressing an endoplasmic reticulum localized enzyme. Enhanced cytotoxicity in cells treated under hypoxic conditions correlated with increases in genomic DNA alkylations, with more MC-DNA adducts being formed when the enzyme was expressed closer to its DNA target. No change was observed in the hypoxic/aerobic differential toxicity as a function of enzyme localization. These findings indicate that drug efficacy is increased when the subcellular site of drug activation corresponds to its site of action.     

B16 and CLS91 mouse melanoma cells susceptibility to apoptosis after vincristin treatment in vitro

Tumor cells' chemoresistance is related to the occurrence or lack of apoptosis. Considering the individual choice of cytostatic drugs for cancer patients and tumor cell resistance the research was undertaken. The viability of mouse melanoma B16 and ClS91 cells and apoptosis induction in vitro after treatment with vincristin was examined. In the future, this kind of study may play an important role in the efficient choice of drug dose and in limiting the side-effects in patients treated with vincristin. Determination of vincristin's influence on cell proliferation kinetics, cell cycle progression based on DNA content and percentage of apoptosis and necrosis in B16 and ClS91 cells, was the object of the present study. The number of viable B16 and ClS91 cells was estimated by flow cytometry analysis. Apoptotic cells were detected using the annexin V-FITC test. Flow cytometric measurement allowed for simultaneous quantitation analysis of four cell subpopulations in the investigated probes. The subpopulations were viable, apoptotic, secondary necrotic and necrotic cells. After adding vincristin into B16 and ClS91 cultures, it was revealed that about 94% ClS91 cells and 45% B16 cells died in the apoptotic way. ClS91 melanoma cells were more sensitive to vincristin treatment than the B16 cell line. The EC50 value for the B16 line was 39.8 microM and for ClS91 was 16.7 microM. Cell cycle was established on the basis of DNA cell content after staining cells with propidium iodide and analysed by flow cytometry. Vincristin induced both B16 and ClS91 cell lines arrest in G2/M cell cycle phase. It was found a correlation between apoptosis occurrence in the melanoma cells and vincristin resistance.     

Bosutinib in Combination With the Aromatase Inhibitor Exemestane: A Phase II Trial in Postmenopausal Women With Previously Treated Locally Advanced or Metastatic Hormone Receptor‐Positive/HER2‐Negative Breast Cancer

Background.

Bosutinib is an oral, selective Src/Abl tyrosine kinase inhibitor with activity in breast cancer (BC). We evaluated bosutinib plus exemestane as second-line therapy in previously treated hormone receptor-positive (HR+) locally advanced or metastatic BC.

Methods.

This was a phase II study with patients enrolled in a single-arm safety lead-in phase. Patients receiving bosutinib at 400 mg or 300 mg/day (based on toxicity) plus exemestane at 25 mg/day were monitored for adverse events (AEs) and dose-limiting toxicities for 28 days, and initial efficacy was assessed. After the lead-in and dose-determination phase, randomized evaluation of combination therapy versus exemestane was planned.

Results.

Thirty-nine of 42 patients (93%) experienced treatment-related AEs including diarrhea in 28 (67%) and hepatotoxicity in 11 (26%); overall serious treatment-related AEs were recorded in 4 (10%). No liver toxicity met Hy’s law criteria. Dose-limiting toxicities occurred in 5 of 13 patients receiving 400 mg (38%) and 3 of 26 patients receiving 300 mg (12%) of bosutinib; all resolved on treatment discontinuation. One patient (300 mg/day) achieved confirmed partial response; three (400 mg/day, n = 2; 300 mg/day, n = 1) maintained stable disease for >24 weeks; a best response of progressive disease occurred in 15 of 42 patients (36%). Median progression-free survival was 12.3 weeks (80% confidence interval: 11.0–15.6).

Conclusion.

The risk-benefit profile of bosutinib at 300 mg/day plus exemestane resulted in early study termination before the randomized portion. Alternative bosutinib regimens merit investigation in BC.     

Axillary sentinel node and tumour-related factors associated with non-sentinel node involvement in breast cancer

BACKGROUND: After completion of axillary dissection, many breast cancer patients with axillary sentinel nodal involvement are found to have regional disease limited to the sentinel nodes. These patients are exposed to the morbidity of axillary clearance without any expected therapeutic benefit. METHODS: Sentinel node biopsy was performed either with Patent blue dye or with a combined dye, radiocolloid and gamma-probe-guided method involving peritumoral tracer administration. For a series of 150 consecutive patients with involved axillary sentinel nodes and axillary dissection, factors associated with non-sentinel nodal involvement were analysed in a multivariate analysis based on logistic regression with the use of fractional polynomials. RESULTS: The following variables were found to be potentially associated with non-sentinel node metastases: tumour size, sentinel node metastasis size, number of examined sentinel nodes, percentage of involved sentinel nodes (the latter two were found to be significant only when in combination), and extracapsular perinodal spread. CONCLUSIONS: Isolated tumour cells and micrometastases in axillary sentinel nodes carry a low risk of non-sentinel node metastasis. The risk of metastasis to further echelon nodes is higher with macrometastases, especially if there is extracapsular growth and the proportion of involved sentinel nodes is high.     

Increased rates of chromosome breakage in BRCA1 carriers are normalized by oral selenium supplementation.

Women who are born with constitutional heterozygous mutations of the BRCA1 gene face greatly increased risks of breast and ovarian cancer. The product of the BRCA1 gene is involved in the repair of double-stranded DNA breaks and it is believed that increased susceptibility to DNA breakage contributes to the cancer phenotype. It is hoped therefore that preventive strategies designed to reduce chromosome damage will also reduce the rate of cancer in these women. To test for increased mutagenicity of cells from BRCA1 carriers, the frequency of chromosome breaks was measured in cultured blood lymphocytes following in vitro exposure to bleomycin in female BRCA1 carriers and was compared with noncarrier relatives. The frequency of chromosome breaks was also measured in BRCA1 carriers following oral selenium supplementation. Carriers of BRCA1 mutations showed significantly greater mean frequencies of induced chromosome breaks per cell than did healthy noncarrier relatives (0.58 versus 0.39; P < 10(-4)). The frequency of chromosome breaks was greatly reduced following 1 to 3 months of oral selenium supplementation (mean, 0.63 breaks per cell versus 0.40; P < 10(-10)). The mean level of chromosome breaks in carriers following supplementation was similar to that of the noncarrier controls (0.40 versus 0.39). Oral selenium is a good candidate for chemoprevention in women who carry a mutation in the BRCA1 gene.     

Peptidoglycan N-acetylglucosamine deacetylase,a putative virulence factor in Streptococcus pneumoniae

Many glucosamine residues of the pneumococcal peptidoglycan (PG) are not acetylated, which makes the PG resistant to lysozyme. A capsular type III mutant with an inactivated pgdA gene (encoding the peptidoglycan N-acetylglucosamine deacetylase A) became hypersensitive to exogenous lysozyme and showed reduced virulence in the intraperitoneal mouse model.     

Gene Polymorphisms of Novel Immunotolerant Molecule BTLA: Distribution of Alleles,Genotypes and Haplotypes in Polish Caucasian Population

B and T lymphocyte attenuator (BTLA) is one of the members of immunoglobulin superfamily which, like CTLA-4 and PD-1, is involved in down regulation of immune response. Despite the important role of BTLA in maintaining immune homeostasis, relatively little studies were devoted to the relationship of polymorphisms in the gene encoding BTLA with susceptibility to autoimmune disease and cancer. Moreover, all published works were done in Asian populations. BTLA gene is located on chromosome 3 in q13.2 and consists of five exons. The aim of this study was to investigate the alleles, genotypes and haplotypes frequency of selected BTLA gene polymorphisms in Caucasian population originating from Poland. For this study, the single-nucleotide polymorphisms (SNPs) were chosen on the basis of literature data. Additionally, the tag dSNP under linkage equilibrium r ² > 0.8 and available at the National Center for Biotechnology Information (NCBI) for Caucasian population of rare alleles at a frequency greater than 5 % have been chosen using the NCBI database. The ten BTLA SNPs investigated were: rs1844089, rs2705535, rs9288952, rs9288953, rs1982809, rs2633580, rs2705511, rs2705565, rs76844316, rs16859633. For all SNPs selected on the basis of literature data the significantly different distributions of genotypes between Asian and Caucasian populations were observed.