Deletion of myosin VI causes slow retinal optic neuropathy and age-related macular degeneration (AMD)-relevant retinal phenotype.

The unconventional myosin VI, a member of the actin-based motor protein family of myosins, is expressed in the retina. Its deletion was previously shown to reduce amplitudes of the a- and b-waves of the electroretinogram. Analyzing wild-type and myosin VI-deficient Snell's Waltzer mice in more detail, the expression pattern of myosin VI in retinal pigment epithelium, outer limiting membrane, and outer plexiform layer could be linked with differential progressing ocular deficits. These encompassed reduced a-waves and b-waves and disturbed oscillatory potentials in the electroretinogram, photoreceptor cell death, retinal microglia infiltration, and formation of basal laminar deposits. A phenotype comprising features of glaucoma (neurodegeneration) and age-related macular degeneration could thus be uncovered that suggests dysfunction of myosin VI and its variable cargo adaptor proteins for membrane sorting and autophagy, as possible candidate mediators for both disease forms.

Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes.

Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.

In vivo analysis of cone survival in mice.

PURPOSE: To identify individual cone photoreceptors in a transgenic mouse line in vivo based on selective expression of green fluorescent protein (GFP) using cSLO (confocal scanning laser ophthalmoscopy) and to use this approach to monitor cone cell fate in mouse models of retinal degeneration. METHODS: Transgenic mice expressing GFP under the control of a red-green opsin promoter (RG-GFP mice) were analyzed in vivo with respect to GFP expression in cone cells using cSLO and functional integrity using electroretinography (ERG). Histology was performed to correlate the pattern of GFP expression with light microscopic data. Longitudinal monitoring of cone survival was evaluated in crossbreds of RG-GFP mice with cpfl1 and Rpe65(-/-) mutant mice, respectively. RESULTS: The authors found that RG-GFP transgenic mice had a stable GFP expression that did not interfere with retinal function up to at least 3 months of age. Thus, a longitudinal analysis of cone degeneration in individual RG cpfl1 and RG Rpe65(-/-) cross-bred mice in vivo was successfully performed and demonstrated distinct time frames of cone survival in the particular mouse model. CONCLUSIONS: Monitoring GFP expression in cone photoreceptor cells, such as in the RG-GFP mouse, is a promising in vivo approach for the analysis of cone survival in mice.

Prognostic significance of RASSF1A promoter methylation on survival of non-small cell lung cancer patients treated with gemcitabine.

The epigenetic inactivation of genes plays an important role in lung cancer. We have investigated the methylation status of the promoter region of seven genes (APC1A, DAPK, FHIT, p14(ARF), p16(INK4a), RARbeta, RASSF1A) in serum DNA of NSCLC patients. The objective of our study was to reveal the influence of such alterations on overall survival. Blood samples were drawn pretherapeutically. Genomic DNA was purified from serum, treated with sodium bisulfite and hypermethylation was detected by a nested methylation-specific PCR in a group of 92 patients with histologically confirmed stage IIIB and IV NSCLC. All patients received gemcitabine first-line alone or in combination with other drugs. The vast majority (n=87) showed at least one epigenetic alteration. The methylation frequencies of individual genes varied between 25.9 and 47.3%. The hypermethylation status of none of the genes had a significant influence on median overall survival of the total population. In contrast, patients with a methylated RASSF1A gene who showed a partial response survived significantly longer (33.6+/-10.4 month) compared to those with a wild-type allele (12.9+/-4.7 month, P=0.0045). This effect became even more pronounced in combination with p14(ARF) (P=0.0004). This difference was not seen in patients with stable or progressive disease. A multivariate analysis confirmed that RASSF1A methylation was an independent prognostic factor. Our results show that the hypermethylation frequency of single genes and the accumulation of epigenetic alterations in individual samples of NSCLC patients may vary considerably. Molecular parameters such as hypermethylation of RASSF1A or p14(ARF) may be useful prognostic markers in subpopulations.

Promoter methylation of RASSF1A, RARbeta and DAPK predict poor prognosis of patients with malignant mesothelioma.

Hypermethylation occurs frequently in neoplastic cells and affects tumorigenesis. Malignant mesothelioma is an aggressive cancer developing in the thoracic cavity and patients have a rather bad prognosis. Our goal was to determine epigenetic alterations of a series of genes and to analyse the potential correlation of such changes with overall survival. We have analysed the methylation status of the promoter region of nine genes in serum DNA of mesothelioma patients by a nested methylation-specific PCR. Modest methylation frequencies were detected for APC1A (14.3%), RASSF1A (19.5%) and DAPK (20.0%) while hypermethylation of E-cadherin (71.4%) and FHIT (78.0%) occurred at a high incidence. Intermediate values were seen for p16(INK4a) (28.2%), APC1B (32.5%), p14(ARF) (44.2%) and RARbeta (55.8%). The methylation status of none of the single genes significantly influenced prognosis. In contrast, combining RARbeta with either DAPK or RASSF1A showed a significantly shorter overall survival of those patients who had both genes methylated compared to those with only one or no epigenetic alteration (P=0.025 and 0.040, respectively). Similarly, the combination of all three genes revealed a worse prognosis for patients with double or triple methylations compared to the group which had only one or no gene methylated (P=0.028). Our results support the idea that the prognostic value of a combination of epigenetic alterations is superior to the impact of an individual gene alone on overall survival.

[Aberrant promoter methylation of tumor suppressor genes in serum from lung cancer patients: frequency and correlation with clinicopathological characteristics]. / Navika slopinanciu metilintu genu daznis plauciu veziu serganciuju kraujo serume bei ju rysys su klinikinemis plauciu vezio charakteristikomis.

OBJECTIVE: The aim of this study was to determine aberrant promoter methylation of tumor suppressor genes in genomic serum DNA from lung cancer patients and to evaluate the association between methylation of genes and clinicopathological characteristics. MATERIALS AND METHODS: Genomic serum DNA from 46 lung cancer patients and 14 healthy control persons was examined. Nested methylation-specific PCR approach was used for detection of methylated genes in serum. RESULTS: Aberrant promoter methylation in serum from lung cancer patients was detected in 2.3% (1/43) for O6-methylguanine-DNA-methyltransferase, in 11.1% (5/45) for p16INK4a, in 41.3% (19/46) for retinoic acid receptor beta, in 4.5% (2/44) for RAS association domain family 1A, in 40.9% (18/44) for fragile histidine triad, in 34.9% (15/45) for p14(ARF), in 6.5% (3/46) for adenomatous polyposis coli 1A, in 78.1% (25/32) for Ecad, in 5.7% (2/35) for adenomatous polyposis coli 1B, in 0% (0/36) for death associated protein kinase. None of the death associated protein kinase, O6-methylguanine-DNA-methyltransferase, p16INK4a, fragile histidine triad or adenomatous polyposis coli 1A promoter regions were positive for methylation in serum DNA from healthy persons. A total of 78.3% of the samples from lung cancer patients had methylation in at least one of the genes tested. The methylation status of fragile histidine triad was associated with that of p14ARF(p=0.021), as was retinoic acid receptor beta--with that of adenomatous polyposis coli 1A (p=0.04). Methylation of adenomatous polyposis coli 1A was detected more frequently in tumor with pleura involvement (p=0.079), retinoic acid receptor beta was observed more frequently in undifferentiated tumor than in better-differentiated tumor (p=0.029). The methylation status of RAS association domain family 1A and fragile histidine triad showed a tendency to be associated with an advanced tumor size. In addition, tumors with an advanced pathological stage showed epigenetic alteration of the RAS association domain family 1A promoter with a higher frequency. The methylation index was also associated with an advanced tumor size (p=0.033). CONCLUSION: Approximately 80% of the samples from lung cancer patients had methylation of the tumor suppressor gene and it might be associated with more aggressive tumor. Detection of epigenetic alterations in serum may provide basis for the early and noninvasive lung cancer diagnosis and customized therapy.