In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells.

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS(61K) in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation.

Live-born trisomy 22: patient report and review.

Trisomy 22 is a common trisomy in spontaneous abortions. In contrast, live-born trisomy 22 is rarely seen due to severe organ malformations associated with this condition. Here, we report on a male infant with complete, non-mosaic trisomy 22 born at 35 + 5 weeks via caesarean section. Peripheral blood lymphocytes and fibroblasts showed an additional chromosome 22 in all metaphases analyzed (47,XY,+22). In addition, array CGH confirmed complete trisomy 22. The patient's clinical features included dolichocephalus, hypertelorism, flattened nasal bridge, dysplastic ears with preauricular sinuses and tags, medial cleft palate, anal atresia, and coronary hypospadias with scrotum bipartitum. Essential treatment was implemented in close coordination with the parents. The child died 29 days after birth due to respiratory insufficiency and deterioration of renal function. Our patient's history complements other reports illustrating that children with complete trisomy 22 may survive until birth and beyond.

Distribution of (TTAGGG)n telomeric sequences in karyotypes of the Xenopus species complex.

The chromosomal distribution of the conserved vertebrate telomeric (TTAGGG)(n) sequence was studied by fluorescence in situ hybridization (FISH) in four Xenopus species and the triploid Silurana tropicalis. As expected, hybridization signals were observed at the distal ends of every chromosome in all species. In addition, the hybridization pattern demonstrates varied organization of (TTAGGG)(n) sequences in the different karyotypes. In X. borealis and X. muelleri hybridization signals intensely labeled one end of a homologous chromosome pair that coincides with the sites containing ribosomal RNA gene clusters. The karyotype of X. clivii remarkably differs from other Xenopus karyotypes in displaying numerous interstitial telomeric sites (ITS). C-banding analysis shows that the non-telomeric sites appear to correspond to the interstitially located constitutive heterochromatin. This suggests that interstitial telomeric sites in X. clivii do not necessarily represent the relic of ancestral telomeres resulting from the fusion of chromosomes, but their occurrence is due to the fact that (TTAGGG)(n) repeat arrays may be a constituent of highly repetitive DNA.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

Fanconi anemia: causes and consequences of genetic instability.

Fanconi anemia (FA) is a rare recessive disease that reflects the cellular and phenotypic consequences of genetic instability: growth retardation, congenital malformations, bone marrow failure, high risk of neoplasia, and premature aging. At the cellular level, manifestations of genetic instability include chromosomal breakage, cell cycle disturbance, and increased somatic mutation rates. FA cells are exquisitely sensitive towards oxygen and alkylating drugs such as mitomycin C or diepoxybutane, pointing to a function of FA genes in the defense against reactive oxygen species and other DNA damaging agents. FA is caused by biallelic mutations in at least 12 different genes which appear to function in the maintenance of genomic stability. Eight of the FA proteins form a nuclear core complex with a catalytic function involving ubiquitination of the central FANCD2 protein. The posttranslational modification of FANCD2 promotes its accumulation in nuclear foci, together with known DNA maintenance proteins such as BRCA1, BRCA2, and the RAD51 recombinase. Biallelic mutations in BRCA2 cause a severe FA-like phenotype, as do biallelic mutations in FANCD2. In fact, only leaky or hypomorphic mutations in this central group of FA genes appear to be compatible with life birth and survival. The newly discovered FANCJ (= BRIP1) and FANCM (= Hef ) genes correspond to known DNA-maintenance genes (helicase resp. helicase-associated endonuclease for fork-structured DNA). These genes provide the most convincing evidence to date of a direct involvement of FA genes in DNA repair functions associated with the resolution of DNA crosslinks and stalled replication forks. Even though genetic instability caused by mutational inactivation of the FANC genes has detrimental effects for the majority of FA patients, around 20% of patients appear to benefit from genetic instability since genetic instability also increases the chance of somatic reversion of their constitutional mutations. Intragenic crossover, gene conversion, back mutation and compensating mutations in cis have all been observed in revertant, and, consequently, mosaic FA-patients, leading to improved bone marrow function. There probably is no other experiment of nature in our species in which causes and consequences of genetic instability, including the role of reactive oxygen species, can be better documented and explored than in FA.

Human puromycin-sensitive aminopeptidase: cloning of 3' UTR, evidence for a polymorphism at a.a. 140 and refined chromosomal localization to 17q21.

Puromycin-sensitive aminopeptidase is a predominantly cytoplasmatic zinc-dependent exopeptidase. Its physiological function is not known to date. Here we report data on tissue distribution, a polymorphism within the coding region and the complete 3' UTR. The gene (NPEPPS alias PSA) was physically mapped to chromosome 17q21.2-->q21.32 using fluorescence in situ hybridization.

Genomic plasticity and melanoma formation in the fish Xiphophorus.

Melanoma formation in certain interspecific hybrids of the genus Xiphophorus (Teleostei: Poeciliidae) is associated with the overexpression of the Xmrk receptor tyrosine kinase oncogene. The Xmrk oncogene arose by duplication of the pre-existing Xmrk protooncogene in a highly unstable subtelomeric region of the X and Y sex chromosomes undergoing frequent rearrangements including duplications, deletions, amplifications, and transpositions. Some of these rearrangements are likely to be responsible for the overexpression of the Xmrk oncogene in melanoma. The oncogene itself is very unstable in Xiphophorus and is frequently removed by deletion or disrupted by transposable elements. The Xmrk oncogene region displays a high concentration of retroelements not observed in the corresponding Xmrk protooncogene region. Particularly, a retrovirus long terminal repeat-like sequence was amplified in the proximity of the Xmrk oncogene. Additional genes, some of them also duplicated copies, were detected in this region and might be involved in modulating the melanoma's phenotype.

hKCNN3 which maps to chromosome 1q21 is not the causative gene in periodic catatonia, a familial subtype of schizophrenia.

The human calcium-activated potassium channel gene (hKCNN3, hSKCa3) contains two tandemly arranged, multiallelic CAG repeats located in exon 1 which result in short to moderate polyglutamine stretches of unknown functional significance. Case-control and family-based association studies suggested an association of hKCNN3 repeats with susceptibility for schizophrenia. Twelve multiplex pedigrees with periodic catatonia, a schizophrenia subtype with major gene effect and patterns of anticipation, were genotyped using the multiallelic hKCNN3 repeat polymorphism. Using a dominant model of inheritance with sex- and age-dependent penetrance classes, cumulative results showed exclusion of linkage of hKCNN3 to periodic catatonia under the assumption of genetic homogeneity with lod score of -48.01 at zero recombination fraction. Our results provide evidence that hKCNN3 is not the causative gene in the familial schizophrenia subtype of periodic catatonia. By fluorescent in situ hybridization we confirmed the assignment of hKCNN3 to chromosome 1q21 near the heterochromatin region. Linkage mapping showed segregation with marker D1S498 (theta = 0.05) and placed hKCNN3 in the genetic linkage map in a cluster of genes near the centromeric region of chromosome 1.

Amplification of a long terminal repeat-like element on the Y chromosome of the platyfish, Xiphophorus maculatus.

The platyfish (Xiphophorus maculatus), in which sex chromosomes are evident from stable and predictable inheritance of sex, is one of the best-studied lower vertebrates with respect to sex determination. In order to identify the structural equivalent for this in the karyotype, which does not contain heteromorphic pairs of chromosomes, two sex-linked molecular probes were used for fluorescent in situ hybridization analysis. One probe, derived from the melanoma oncogene locus ONC-Xmrk, stained both the X and the Y chromosome. This cytogenetic analysis mapped the sex-determining locus to the subtelomeric region of a medium-sized telocentric chromosome. Another probe, a repetitive element (XIR), specifically labeled the Y chromosome in metaphase spreads and in interphase nuclei. The sex chromosomes of X. maculatus can be considered to be at an early stage of evolution of gonosomes. Expansion of the XIR repeat is obviously one of the earliest of the molecular events that lead to divergence of the Y chromosome and recombinational isolation of the sex-determining locus.

Chromosomal localization of the genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma, and VIM in chicken by fluorescence in situ hybridization.

Six structural genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma and VIM were mapped in the chicken by fluorescence in situ hybridization (FISH) using genomic and cDNA clones as probes. The genes were found to be located on four different macrochromosomes: chromosome 1 (IFNG and FABP), chromosome 2 (VIM and ALDH), chromosome 3 (BMP2) and a smaller macrochromosome, most probably chromosome 7 (RXRG). With the exception of IFNG none of the newly mapped sites corresponds to known orthologous regions between chicken and human chromosomes.

Unusual triploid males in a microchromosome-carrying clone of the Amazon molly, Poecilia formosa.

The Amazon molly, Poecilia formosa, is an all-female fish of hybrid origin which reproduces by gynogenesis, i.e. it depends on sperm of males of closely related species to trigger parthenogenetic development of the embryo. Therefore the offspring is clonal and identical to the mother. In rare cases the exclusion mechanism fails and paternal introgression occurs. This may result either in triploid offspring - if the whole haploid chromosome set of the sperm fuses with the diploid egg nucleus - or in siblings with microchromosomes - if only subgenomic amounts of paternal DNA are included. In one of our diploid, microchromosome-carrying laboratory stocks we observed eight triploid individuals which all developed into males. We investigated the mitotic and meiotic chromosomes, the synaptonemal complex (SC), and sperm production of these males, and compared them to males of the gonochoristic parental species (P. latipinna and P. mexicana) and their hybrids. This comparison revealed that P. formosa males are functional males with reduced effective fertility. They show a deviation from the typical 23 bivalents in the synaptonemal complexes as well as in diakinesis due to the triploid state. They produced offspring but only with gynogenetic Amazon molly females. This shows that the probably aneuploid sperm from P. formosa males can trigger parthenogenetic development of unreduced eggs.

Sex chromosome linkage of chicken and duck type I interferon genes: further evidence of evolutionary conservation of the Z chromosome in birds.

Type I interferons (IFNs) are a family of proteins that are predominantly expressed in response to viral infection. Two serologically distinct forms of type I IFN, designated ChIFN1 and ChIFN2, have recently been recognized in the chicken. ChIFN1 is encoded by a cluster of ten or more intronless genes, whereas ChIFN2, whose primary sequence is 57% identical, is encoded by a single intronless gene. By fluorescence in situ hybridization we now demonstrate that the genes for ChIFN1 and ChIFN2 are all located on the short arm of the chicken Z chromosome. This assignment was confirmed by results that showed that DNA from male (ZZ) chickens yielded approximately twofold stronger Southern blot signals with ChIFN1 and ChIFN2 hybridization probes than DNA from females (ZW). Attempts to determine differences in IFN production between male and female chickens failed owing to a high degree of variation in virus-induced IFN expression between individuals of both sexes. Sex linkage of IFN genes was also observed in domestic ducks: fluorescence in situ hybridization of duck metaphase chromosomes with a duck type I IFN probe was confined to the terminal region of the long arm of the Z chromosome. Thus, in contrast to mammals, which have their IFN genes on autosomes, birds have the type I IFN genes on the sex chromosome.

Susceptibility to the development of pigment cell tumors in a clone of the Amazon molly, Poecilia formosa, introduced through a microchromosome.

The Amazon molly Poecilia formosa is a gynogenetic fish that reproduces through the development of ameiotic diploid eggs triggered by insemination by males of related species without following karyogamie. This leads to clonal offspring. In rare cases, however, this gynogenesis is leaky, and paternal DNA in the form of small supernumerary chromosomes is included into the maternal genome. We have obtained a clone where one such microchromosome contains a pigmentary locus, resulting in macromelanophore pigmentation of the carrier. Approximately 5% of these fish spontaneously develop exophytic nodular or papillomatous pigment cell tumors. The tumors display considerable differences with respect to growth characteristics and invasiveness, despite the genetic uniformity of the affected animals. Following transplantation to syngeneic hosts, a remarkable clonal variability was observed. Oncogenes that are involved in tumorigenesis in hereditary melanoma of the closely related fish Xiphophorus appear not to be instrumental for induction of the P. formosa pigment cell tumors. Moreover, a new genetic locus is defined that mediates susceptibility to pigment cell tumor development and leads to transformation of chromatoblasts.

Assignment of the chicken MAX gene to chromosome 5p by fluorescence in situ hybridization.

It has been shown that the protein encoded by the MAX gene plays an important role in the physiological activity of Myc oncoproteins. In this study, we determined the chromosome location of the chicken MAX gene via fluorescence in situ hybridization. Hybridization of two biotinylated cloned fragments of 5.7 kb and 12.0 kb derived from the chicken MAX locus localized the gene to chromosome 5p. It is the third gene marker to be assigned to this telocentric macrochromosome. Since the MAX sequence is highly conserved both at the nucleotide and at the amino acid level in a wide range of vertebrate species, our data may provide evidence for the existence of a segmental homology between human and chicken chromosomes.

Clustered organization and conservation of the Xiphophorus maculatus D locus, which includes two distinct gene sequences.

Genomic organization and chromosomal localization of a previously uncharacterized D (Donor) locus in Xiphophorus and Poecilia species was investigated using fluorescence in situ hybridization (FISH) and Southern blot analysis. Part of this region is thought to be involved in the recombination event leading to formation of the Xmrk oncogene and it has recently been shown that this locus includes two different genes, one with high homology to a zinc finger protein of the Krüppel type, and the other an unknown gene with high similarity to a Caenorhabditis elegans gene. FISH to Xiphophorus chromosomes revealed that these two unrelated genes are closely linked and clustered at a unique chromosomal site. Southern blot hybridization patterns suggest that these genes exist in the genome as multiple copies. Furthermore, similar genomic organization profiles seem to prevail among other related fish. In particular, our FISH experiments reveal the existence of a conserved homologous chromosomal segment harboring the zinc finger protein sequence in several poeciliid fish.

The Xmrk oncogene promoter is derived from a novel amplified locus of unusual organization.

Hereditary melanoma in Xiphophorus hybrids is caused by the receptor tyrosine kinase Xmrk. Tumor formation is initiated by overexpression of the Xmrk gene, apparently because of insufficient transcriptional control in the melanocytic lineage of hybrid fish. The oncogenic Xmrk resulted from gene duplication and nonhomologous recombination of the corresponding Xmrk proto-oncogene during evolution. By this event Xmrk was translocated downstream of the promoter of another gene, D (for Donor). This raised the question whether both the Xmrk oncogene and D share similar transcriptional control elements. Studies on the genomic organization of D showed that this gene is amplified in the Xiphophorus genome, presumably with all copies clustered on a single chromosome. Surprisingly, at least two completely different, tightly linked genes are included in the amplified segment. We find a ubiquitously expressed zinc finger gene of the krüppel type, followed by a previously unknown gene, which was the partner of the Xmrk proto-oncogene in the recombination generating the Xmrk oncogene. The nucleotide sequence predicts a gene product with very high amino acid similarity to a hypothetical Caenorhabditis elegans protein. The expression pattern is unrelated to that of the Xmrk oncogene suggesting that despite extended sequence homology a new type of promoter was created by this rearrangement.

Mammalian sex chromosomes. II. Pairing and alignment of the X and Y chromosomes of the pygmy mouse, Mus dunni.

In the pygmy mouse, Mus dunni, the entire Y chromosome and the short arm of the X and distal region of its long arm are constitutively heterochromatic. Different banding studies on somatic chromosomes revealed the GC nature of the distally located heterochromatin of the long arms of both the X and Y chromosomes. The short arm of the X and the rest of the Y are AT-rich. During meiosis, the long arms of the X and Y paired extensively, sometimes more than half of the Y pairing with the X. This observation is in disagreement with that of Pathak and Hsu (1976) who reported end-to-end pairing between the long arm of the X and the short arm of the Y. The orientation observed by us is favourable to a successful meiotic recombination but whether this takes place remains to be demonstrated.

Mammalian sex chromosomes. I. Cytological changes in the chiasmatic sex chromosomes of the male musk shrew, Suncus murinus.

The X and Y chromosomes of the musk shrew are the two largest in the complement and they regularly form a single chiasma during meiosis. This chiasma is located in the short arms of the X and Y, both of which show partial C-banding at meiosis. The in vitro incorporation of 5-bromodeoxyuridine/tritiated thymidine during late S reveals that the non-C-band region of the Y finishes replication later than the C-band positive heterochromatin. During meiosis, the sex bivalent opens out early in pachytene to reveal a single chiasma which persists until late metaphase-I. In surface-spread, silver-stained meiocytes, the sex bivalent morphology changes from a phase of extensive pairing to one which includes a visible chiasma through a brief diffuse stage. Observations on C-banded meiocytes show a shift in the sex pair from a C-band positive to a negative state as compared to their corresponding somatic pattern. Comparable changes are also observed in the sex bivalents of other mammals which undergo a chiasmatic exchange. This suggests that in addition to pairing homology, an alteration in the chromatin configuration may be necessary for crossing over to occur between the sex chromosomes.

Identification and patterns of synapsis of the autosomally translocated Y-chromosome of the Indian mongoose, Herpestes auropunctatus (Hodgson).

The multiple sex chromosome system, X1X2Y male/X1X1X2X2 female, in the small Indian mongoose, Herpestes auropunctatus, results from a translocation of a part of Y chromosome to an autosome. It is not possible to distinguish the autosome which harbours the Y chromosome element in the somatic complement. By employing the surface-spreading technique to prophase I meiocytes we have identified the region to which the Y chromosome has been translocated as the short arm of chromosome 9 which is a subtelocentric chromosome. This Y chromosome component lacks heterochromatin and no sex vesicle is organised during meiotic prophase. This suggests to us that Y heterochromatin in mammals may be required for the production of a sex vesicle.