High level of chromosome 15 aneuploidy in head and neck squamous cell carcinoma lesions identified by FISH analysis: limited value of beta2-microglobulin LOH analysis.

In cancer research, loss of heterozygosity (LOH), defined by microsatellite markers, is frequently used in the identification of gene loss. Especially, genomic alterations in the human leukocyte antigen (HLA) genes and the beta2-microglobulin (beta2m) gene on chromosome 15 are of interest regarding their function in the immune system. Because LOH analysis detects any allelic imbalance and not just allelic loss, we evaluated the LOH analysis in 11 head and neck squamous cell carcinoma (HNSCC) lesions using fluorescence in situ hybridization (FISH). The 11 tumors were selected out of 53 HNSCC lesions based upon beta2m LOH analysis and beta2m expression. Centromere 1 and 15 FISH were developed to determine the chromosome 15 copy number. Sequence-based mutation analysis of beta2m was conducted on tumors without beta2m expression; no mutations in the coding sequences were found. For five HNSCC lesions with LOH and beta2m expression, centromere 15 FISH indicated gain rather than loss. In the majority of the 11 HNSCC lesions, FISH showed centromere 1 and 15 heterogeneity throughout the tumor. Moreover, FISH indicated a more complex chromosome 1 and 15 distribution than could be concluded from microsatellite LOH analysis. Our results show that microsatellite LOH analysis does not represent the beta2m gene copy number and support the results obtained from comparative genomic hybridization (CGH) studies. Conclusions on genomic alterations in tumors cannot be based on LOH data only but depend on the results of immunohistochemical staining, FISH, and CGH.

Alkyl-dihydroxyacetonephosphate synthase. Presence and role of flavin adenine dinucleotide.

Alkyl-dihydroxyacetonephosphate synthase is a peroxisomal enzyme involved in ether lipid synthesis. It catalyzes the exchange of the acyl chain in acyl-dihydroxyacetonephosphate for a long chain fatty alcohol, yielding the first ether linked intermediate, i.e. alkyl-dihydroxyacetonephosphate, in the pathway of ether lipid biosynthesis. Although this reaction is not a net redox reaction, the amino acid sequence of the enzyme suggested the presence of a flavin adenine dinucleotide (FAD)-binding domain. In this study we show that alkyl-dihydroxyacetonephosphate synthase contains an essential FAD molecule as cofactor, which is evidenced by fluorescence properties, UV-visible absorption spectra and the observation that the enzyme activity is dependent on the presence of this cofactor in a coupled in vitro transcription/translation assay. Furthermore, we could demonstrate that the FAD cofactor directly participates in catalysis. Upon incubation of the enzyme with the substrate palmitoyl-dihydroxyacetonephosphate, the flavin moiety is reduced, indicating that in this initial step the substrate is oxidized. Stopped flow experiments show that the reduction of the flavin moiety is a monophasic process yielding a oxygen stable, reduced enzyme species. Upon addition of hexadecanol to the reduced enzyme species, the flavin moiety is efficiently reoxidized. A hypothetical reaction mechanism is proposed that is consistent with the data in this paper and with previous studies.