Cisplatin-resistant neuroblastoma cells express enhanced levels of epidermal growth factor receptor (EGFR) and are sensitive to treatment with EGFR-specific toxins

PURPOSE: Neuroblastomas frequently show expression of the epidermal growth factor receptor (EGFR) and may therefore be susceptible to EGFR-targeted therapies. Here, EGFR expression and functionality was investigated in parental chemosensitive neuroblastoma cell lines (UKF-NB-3, IMR-32, NLF, SH-SY5Y) and their cisplatin-resistant sublines (UKF-NB-3(r)CDDP(1000), IMR-32(r)CDDP(1000), NLF(r)CDDP(1000), and SH-SY5Y(r)CDDP(500)). Moreover, the EGFR antibody cetuximab, the EGFR tyrosine kinase inhibitor Tyrphostin B46, and recombinant EGFR-targeted toxins were investigated for their influence on the viability and growth of neuroblastoma cells. EXPERIMENTAL DESIGN: EGFR expression and function was measured by flow cytometry or Western blot. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was examined by immunostaining for active caspase-3 or cleaved poly(ADP-ribose) polymerase. Cellular binding of FITC-labeled immunotoxins was studied by flow cytometry, and cellular uptake was studied by confocal laser scanning microscopy. RESULTS: The EGFR-targeted antibody and growth factor toxins scFv(14E1)- Pseudomonas exotoxin A (ETA) and TGF-alpha-ETA exerted anti-cancer effects in neuroblastoma cell lines that were insensitive to cetuximab or EGFR tyrosine kinase inhibitors. Furthermore, adaptation of chemosensitive neuroblastoma cells to cisplatin increased EGFR expression and sensitivity to both recombinant toxins. Treatment of chemosensitive neuroblastoma cells with cisplatin reversibly increased EGFR expression, whereas cisplatin-resistant cells showed enhanced EGFR expression independent of the presence of cisplatin. Combination treatment with scFv(14E1)-ETA or TGF-alpha-ETA and cisplatin exerted significantly improved anticancer effects compared with either single treatment in parental neuroblastoma cells, cisplatin-resistant sublines, and primary cultures. CONCLUSIONS: EGFR-targeted cytotoxic reagents such as scFv(14E1)-ETA and TGF-alpha-ETA represent promising candidates for further development as antineuroblastoma agents, especially in combination with cisplatin.     

The AA genotype of the regulatory BCL2 promoter polymorphism ( 938C>A) is associated with a favorable outcome in lymph node negative invasive breast cancer patients.

PURPOSE: Expression of the antiapoptotic and antiproliferative protein Bcl-2 has been repeatedly shown to be associated with better clinical outcome in breast cancer. We recently showed a novel regulatory (-938C>A) single-nucleotide polymorphism (SNP) in the inhibitory P2 BCL2 gene promoter generating significantly different BCL2 promoter activities. EXPERIMENTAL DESIGN: Paraffin-embedded neoplastic and nonneoplastic tissues from 274 patients (161 still alive after a follow-up period of at least 80 months) with primary unilateral invasive breast carcinoma were investigated. Bcl-2 expression of tumor cells was shown by immunohistochemistry; nonneoplastic tissues were used for genotyping. Both the Bcl-2 expression and the (-938C>A) genotypes were correlated with the patients' survival. RESULTS: Kaplan-Meier curves revealed a significant association of the AA genotype with increased survival (P = 0.030) in lymph node-negative breast cancer patients, whereas no genotype effect could be observed in lymph node-positive cases. Ten-year survival rates were 88.6% for the AA genotype, 78.4% for the AC genotype, and 65.8% for the CC genotype. Multivariable Cox regression identified the BCL2 (-938CC) genotype as an independent prognostic factor for cancer-related death in lymph node-negative breast carcinoma patients (hazard ratio, 3.59; P = 0.032). Immunohistochemical Bcl-2 expression was significantly associated with the clinical outcome of lymph node-positive but not of lymph node-negative breast cancer patients. In lymph node-negative cases, the (-938C>A) SNP was both significantly related with the immunohistochemically determined level of Bcl-2 expression (P = 0.044) and the survival of patients with Bcl-2-expressing carcinomas (P = 0.006). CONCLUSIONS: These results suggest the (-938C>A) polymorphism as a survival prognosticator as well as indicator of a high-risk group within patients with lymph node-negative breast cancer.     

The Vienna functional electrical stimulation system for restoration of walking functions in spastic paraplegia

An eight-channel stimulation system, currently intended for stimulation of lower extremities, was developed and is introduced. The major development goals were easy handling, modularity to make the system easily adaptable for other functional electrical stimulation (FES) applications, and a wide stimulation parameter range for application-specific parameter optimization. For paraplegic stepping, the system worn by the patient consists of 2 four-channel stimulation modules, a central unit holding the battery and circuitry for power management and communication control, a wireless remote control unit, and a palmtop computer as the main control and input device. A software package for Microsoft Windows supports the design and optimization of stimulation sequences in the rehabilitation center. First tests with patients familiar with FES showed smoother movements during stepping and acceptable good handling. In combination with the PC software, the required stimulation sequences could be created in a very short time.     

Cooperative effect of GNB3 825C>T and GPIIIa PI(A) polymorphisms in enhanced platelet aggregation

Introduction: Platelet aggregation contributes to various thrombembolic disorders. Environmental factors affect platelet aggregability but only partially explain the interindividual variability in aggregation. While the platelet glycoprotein IIb/IIIa is involved in the pathogenesis of acute coronary syndromes whereas most platelet activating stimuli act via G Protein coupled receptors we investigated whether the 825C>T polymorphism of the gene GNB3 encoding the G protein β3 subunit together with the platelet glycoprotein (GP) IIIa Pl(A) polymorphism are predictive of platelet aggregability on stimulation with various agonists acting via GPCRs. Materials and methods: Platelet aggregation was measured by turbidometry in 150 non-smoking individuals aged 18–40 years at a density of 2×10⁵ platelets/μl with various agonists according to the method of Born. Genotypes of the GNB3 825C>T and glycoprotein IIb/IIIa PI(A) polymorphisms were determined using Pyrosequencing technology and restriction analysis. All functional studies were completed within 3 h. The data were analysed by Student's t-test for paired data. Results: Low concentrations of agonists resulted in enhanced platelet aggregation in subjects with the GNB3 CC-genotype compared to carriers of a 825T-allele. This effect was further enhanced in carriers of the GPIIIa Pl(A2) allele (2 μM ADP: 42% vs. 19%, p=0.017; 1 μM U-46619: 51% vs. 30%, p=0.03; 5 μM epinephrine: 69% vs. 53%, p=0.025). No significant pattern of aggregation was observed on stratification by GPIIIa genotypes alone. Conclusions: Our findings indicate that two genetic markers contribute synergistically to increased platelet aggregation. This will help to identify patients at increased risk for thrombosis.     

Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats

There is a concern that increased paternal age may be associated with altered fertility and an increased incidence of birth defects in man. In previous studies of aged male rats, we have found abnormalities in the fertility and in the embryos sired by older males. Aging in mammals is associated with alterations in the content and patterns of DNA methylation in somatic cells; however, little is known in regard to germ cells. A systematic search for global and gene-specific alterations of DNA methylation in germ cells and liver of male rats was done. Restriction landmark genomic scanning, a method used to determine specific methylation patterns of CpG island sequences, has revealed a region of the ribosomal DNA locus that is preferentially hypermethylated with age in both spermatozoa and liver. In contrast, all single copy CpG island sequences in spermatozoa and in liver remain unaltered with age. We further demonstrate that a large proportion of rat ribosomal DNA is normally methylated and that regional and site-specific differences exist in the patterns of methylation between spermatozoa and liver. We conclude that patterns of ribosomal DNA methylation in spermatozoa are vulnerable to the same age-dependent alterations that we observe in normal aging liver. Failure to maintain normal DNA methylation patterns in male germ cells could be one of the mechanisms underlying age-related abnormalities in fertility and progeny outcome.     

The study of aberrant methylation in cancer via restriction landmark genomic scanning

Restriction landmark genomic scanning (RLGS) has been used to study DNA methylation in cancer for nearly a decade. The strong bias of RLGS for assessing the methylation state of CpG islands genome wide makes this an attractive technique to study both hypo- and hypermethylation of regions of the genome likely to harbor genes. RLGS has been used successfully to identify regions of hypomethylation, candidate tumor suppressor genes, correlations between hypermethylation events and clinical factors, and quantification of hypermethylation in a multitude of malignancies. This review will examine the major uses of RLGS in the study of aberrant methylation in cancer and discuss the significance of some of the findings.     

Role of radiotherapy in the treatment of motor dysfunction due to metastatic spinal cord compression: comparison of three different fractionation schedules

PURPOSE: The optimum fractionation schedule for radiotherapy (RT) of metastatic spinal cord compression (MSCC) is still debated in the literature. Several reports have compared different fractionation schedules for pain relief. To our knowledge, this retrospective analysis is the first to compare three different schedules for functional outcome. METHODS AND MATERIALS: For posttreatment functional and ambulatory outcome, three schedules, 30 Gy in 10 fractions (n = 93), 37.5 Gy in 15 fractions (n = 80), and 40 Gy in 20 fractions (n = 74), were compared. Motor function was evaluated by a 6-point scale before and at the end of RT and 3, 6, and 12 months later. A multivariate analysis was performed for functional outcome, including fractionation schedule and the three relevant prognostic factors (primary tumor type, time of developing motor deficits before RT, and ambulatory status). RESULTS: No significant difference was observed for posttreatment motor function or ambulatory rates among the three schedules. According to the multivariate analysis, the radiation schedule had no significant impact on functional outcome (p = 0.223) in contrast to the three prognostic factors (p <0.001, p <0.001, and p = 0.012). CONCLUSION: The three fractionation schedules were comparable for functional outcome. The least time-consuming schedule (30 Gy in 10 fractions) should be considered for patients with a markedly reduced life expectancy.     

Mbd4 inactivation increases Cright-arrowT transition mutations and promotes gastrointestinal tumor formation

Mbd4 (methyl-CpG binding domain 4) is a novel mammalian repair enzyme that has been implicated biochemically in the repair of mismatched G-T residues at methylated CpG sites. In addition, the human protein has been shown to interact with the DNA mismatch repair protein MLH1. To clarify the role of Mbd4 in DNA repair in vivo and to examine the impact of Mbd4 inactivation on gastrointestinal (GI) tumorigenesis, we introduced a null mutation into the murine Mbd4 gene by gene targeting. Heterozygous and homozygous Mbd4 mutant mice develop normally and do not show increased cancer susceptibility or reduced survival. Although Mbd4 inactivation did not increase microsatellite instability (MSI) in the mouse genome, it did result in a 2- to 3-fold increase in C-->T transition mutations at CpG sequences in splenocytes and epithelial cells of the small intestinal mucosa. The combination of Mbd4 deficiency with a germ line mutation in the adenomatous polyposis coli (Apc) gene increased the tumor number in the GI tract and accelerated tumor progression. The change in the GI cancer phenotype was associated with an increase in somatic C-->T mutations at CpG sites within the coding region of the wild-type Apc allele. These studies indicate that, although inactivation of Mbd4 does not by itself cause cancer predisposition in mice, it can alter the mutation spectrum in cancer cells and modify the cancer predisposition phenotype.     

Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression

Flap endonuclease (Fen1) is required for DNA replication and repair, and defects in the gene encoding Fen1 cause increased accumulation of mutations and genome rearrangements. Because mutations in some genes involved in these processes cause cancer predisposition, we investigated the possibility that Fen1 may function in tumorigenesis of the gastrointestinal tract. Using gene knockout approaches, we introduced a null mutation into murine Fen1. Mice homozygous for the Fen1 mutation were not obtained, suggesting absence of Fen1 expression leads to embryonic lethality. Most Fen1 heterozygous animals appear normal. However, when combined with a mutation in the adenomatous polyposis coli (Apc) gene, double heterozygous animals have increased numbers of adenocarcinomas and decreased survival. The tumors from these mice show microsatellite instability. Because one copy of the Fen1 gene remained intact in tumors, Fen1 haploinsufficiency appears to lead to rapid progression of cancer.