Thilo Papp

Mutational analysis of the N-ras,p53,p16INK4a,CDK4, and MC1R genes in human congenital melanocytic naevi

Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenesN-ras andCDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genesp53 andp16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in theN-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in theMC1R andp53 genes, respectively. No mutations were found in p16 orCDK4. The activatedN-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.

Human health implications of nanomaterial exposure

Nanotechnology presents countless opportunities to develop new and improved consumer products for the benefit of society. However, as the industrial production and use of nanotechnology products continue to expand at a fast scale, potential human health concerns and ecological safeguards for the environment need to be addressed. Health risk assessment involving different animal species for multi-organ toxicity complimented with molecular investigations in cells is essential for investigating the potential toxic effects of nanomaterials. The purpose of this review is to present the current state of knowledge regarding the potential routes of human exposure to nanomaterials and their biological health effects. Although anthropogenic nanosized particles emitted in the environment are known to produce adverse human health in susceptible populations, much remains to be explored. Exposures can occur from direct exposure or from the use of commercial products made of nanomaterials. Safe manufacturing guidelines for prevention of exposures and recommendations on safe handling and use need to be established on a proactive basis to prevent adverse outcomes.

Lack ofp53 mutations and loss of heterozygosity in non-cultured human melanocytic lesions

In this study we analysed snap-frozen surgical resections of 16 superficial spreading melanomas, 13 nodular malignant melanomas, 2 lentigo maligna melanomas, 1 dysplastic nevus, 1 congenital nevus and 5 normal nevi from 38 patients for point mutations in the humanp53 gene at exons 5–8 by polymerase chain reaction/single-strand conformation polymorphism as well as for loss of heterozygosity ofp53 by restriction-fragment-length polymorphism/polymerase chain reaction in order to determine whetherp53 aberrations are associated with melanoma subtypes. In addition, we analysed six melanoma cell lines for point mutations inp53. Our results revealed the absence of point mutations and loss of heterozygosity in all fresh resected lesions. However, a TAC (Tyr) to TGC (Cys) transition at codon 163 in exon 5 was found in one cell line.

Mutational analysis of the BRAF gene in human congenital and dysplastic melanocytic naevi.

Eighteen congenital melanocytic naevi (CMN) from 17 patients and 18 dysplastic melanocytic naevi (DMN) from 18 patients were screened for mutations in the BRAF oncogene (present study) and the N-ras oncogene (in the course of two foregoing studies) by single-strand conformational polymorphism (SSCP)/sequencing analysis. BRAF mutations were demonstrated in both types of lesion. As a whole, 17 of 18 CMN (94.4%) and five of 18 DMN (27.7%) harboured either BRAF or N-ras mutations. As the BRAF oncogene is frequently found to be mutated in human cutaneous melanomas, it may constitute a risk factor for melanoma formation within CMN and DMN.

Mutational analysis of N-ras , p53 , CDKN2A ( p16 INK4a ), p14 ARF , CDK4 , and MC1R genes in human dysplastic melanocytic naevi

In order to detect possible dysplastic melanocytic naevi (DMN) associated melanoma risk factors and lesion specific differences in the mutation spectrum of dysplastic and congenital melanocytic naevi (CMN), we screened 19 specimens of human sporadic DMN derived from 19 patients for the presence of mutations in five genes, which we had investigated in a former study in 19 CMN1 and which have been reported to be associated with human cutaneous melanoma ( N-ras ,2 p53 ,3 CDKN2A ,4 CDK4 ,5 and MC1R 6,7). DNA was extracted from selected paraffin embedded DMN resection specimens using the QIAamp DNA Mini Kit (Qiagen) according to the recommendations of the supplier. The relative number of atypical melanocytes in the DMN and the histological subtype of the DMN were determined in parallel slides by an experienced dermatologist (Dr Regina Zimmermann) (table 1). View this table: Table 1 N-ras mutations and MC1R variants in human spontaneous dysplastic naevi The screening strategy for the detection of activating point mutations in the oncogenes N-ras and CDK4 as well as for germline sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis, and the screening strategy for the detection of homozygous deletions and point mutations in the tumour suppressor genes p53 and CDKN2A by combined multiplex-PCR/SSCP analysis, have been described previously.1 In order to …

Cytogenetic changes in primary, immortalized and malignant mammalian cells

Some chromosomes in transformed rat cells and somatic cell hybrids fail to display the presence of kinetochore proteins as detected by antikinetochore antibodies. Such chromosomes (K- chromosomes) may constitute a novel mechanism for the genesis of aneuploidy. We have analyzed primary, immortalized and malignant mammalian cells for the presence of kinetochore proteins and micronuclei. Our results suggest a correlation of the K- chromosome and micronucleus frequency with the variability in chromosome number. Upon in situ hybridization with the minor satellite and alpha satellite sequences some K- chromosomes showed a signal. This indicates that the observed lack of kinetochores is not necessarily due to a lack of centromeric DNA. We conclude that dislocated K- chromosomes may become incorporated into micronuclei which are prone to loss. Such events would be associated with the generation of aneuploidy.

Detection of hyperdiploidy and chromosome breakage affecting the 1 (1cen-q12) region in lentigo malignant melanoma (LMM), superficial spreading melanoma (SSM) and congenital nevus (CN) cells in vitro by the multicolor FISH technique

The centric/pericentric region of chromosome 1 (cen-q 2) of human melanoma cells of different stages of carcinogenicity (superficial spreading melanoma (SSM), lentigo malignant melanoma (LMM)) and premalignant precursor lesions (congenital nevus (CN)) were investigated by fluorescence in situ hybridization (FISH) with tandem DNA probes. The pericentric heterochromatin region 1(q12) is large and highly prone to breakage in contrast to the adjacent centromeric region which is much smaller and less prone to such events. All samples of melanoma cells were obtained from patients and cultivated in vitro. LMM cells showed the highest number of breakage events within the 1q12 region (90% of cells). The number of hyperdiploid cells was not increased in comparison to CN cells. In contrast to LMM cells, SSM cells showed a significant increased number of hyperdiploid cells which were mainly tetrasomic for chromosome 1 (P < or = 0.05). The number of chromosome breaks was not significantly increased in this type of melanoma cells. The spontaneous rates of chromosomal breakage and hyperdiploidy is relatively low in CN cells (1.5-2.5% and 3.2-5.8%, respectively) but these frequencies also differ between CN samples from different patients. These results show that the multicolor FISH technique represents a fast and reliable detection method, distinguishing structural and numerical chromosomal alterations in interphase nuclei. This technique is useful as a histological marker to differentiate between specific tumor subtypes and to investigate the relationship between genomic instability and clinopathological parameters (tumor grading and staging).