Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer.

BACKGROUND: The short arm of human chromosome 3 is involved in the development of many cancers including lung cancer. Three bona fide lung cancer tumor suppressor genes namely RBSP3 (AP20 region),NPRL2 and RASSF1A (LUCA region) were identified in the 3p21.3 region. We have shown previously that homozygous deletions in AP20 and LUCA sub-regions often occurred in the same tumor (P < 10-6). METHODS: We estimated the quantity of RBSP3, NPRL2, RASSF1A, GAPDH, RPN1 mRNA and RBSP3 DNA copy number in 59 primary non-small cell lung cancers, including 41 squamous cell and 18 adenocarcinomas by real-time reverse transcription-polymerase chain reaction based on TaqMan technology and relative quantification. RESULTS: We evaluated the relationship between mRNA level and clinicopathologic characteristics in non-small cell lung cancer. A significant expression decrease (> or =2) was found for all three genes early in tumor development: in 85% of cases for RBSP3; 73% for NPRL2 and 67% for RASSF1A (P < 0.001), more strongly pronounced in squamous cell than in adenocarcinomas. Strong suppression of both, NPRL2 and RBSP3 was seen in 100% of cases already at Stage I of squamous cell carcinomas. Deregulation of RASSF1A correlated with tumor progression of squamous cell (P = 0.196) and adenocarcinomas (P < 0.05). Most likely, genetic and epigenetic mechanisms might be responsible for transcriptional inactivation of RBSP3 in non-small cell lung cancers as promoter methylation of RBSP3 according to NotI microarrays data was detected in 80% of squamous cell and in 38% of adenocarcinomas. With NotI microarrays we tested how often LUCA (NPRL2, RASSF1A) and AP20 (RBSP3) regions were deleted or methylated in the same tumor sample and found that this occured in 39% of all studied samples (P < 0.05). CONCLUSION: Our data support the hypothesis that these TSG are involved in tumorigenesis of NSCLC. Both genetic and epigenetic mechanisms contribute to down-regulation of these three genes representing two tumor suppressor clusters in 3p21.3. Most importantly expression of RBSP3, NPRL2 and RASSF1A was simultaneously decreased in the same sample of primary NSCLC: in 39% of cases all these three genes showed reduced expression (P < 0.05).

NotI passporting to identify species composition of complex microbial systems.

We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.

A new approach to genome mapping and sequencing: slalom libraries.

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with approximately 46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of approximately 100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5-10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.

Restriction site tagged (RST) microarrays: a novel technique to study the species composition of complex microbial systems.

We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.

An integrated physical and gene map of the 3.5-Mb chromosome 3p21.3 (AP20) region implicated in major human epithelial malignancies.

To facilitate the identification of tumor suppressor genes in the chromosome 3p21.3-p22 AP20 subregion, we constructed a 3.5-Mb physical and gene map of this segment (between markers D3S4285 and D3S3873) that spans the distance from 124.4cR3000 to 133.5 cR3000 of the GB4 genetic map. We used NotI-linking and -jumping clones, sequence-tagged site PCR marker analysis, and multicolor and fiber fluorescence in situ hybridization to confirm the sequence order and map orientation. An integrated clone contig composed of 5 yeast artificial chromosome, 15 bacterial artificial chromosome, 5 P1 artificial chromosome, and 8 NotI-linking clones provided the physical base of the map. We unequivocally established the order of 28 sequence-tagged sites and 35 genes in the region. Gaps between published bacterial artificial chromosome contigs were determined and covered by our own sequence data. Furthermore, three new genes were isolated, namely the human homologue to the rat Golgi peripheral membrane protein p65, GOLPH5 (GORASP1), the gene for stress-inducible protein, STI2, and the AP20-region gene 1, APRG1. The tumor suppressor gene candidate APRG1 was positioned close to the border of the homozygous deletion in a small cell lung cancer cell line ACC-LC5. Expression analysis with a tissue-specific panel of cDNA revealed seven distinct tissue-specific splice variants (A-G) of the message (size range, 1.0-1.8 kb). Although the gene was expressed at a low level in all tested tissues, comparatively higher expression was detected in pancreas (splice forms B and D), kidney (A) and placenta (B and C). The APRG1 gene encoded a predicted protein of 170 amino acids (isoform B), which had an NH2-terminal part conserved among members of the eukaryotic translation factor 6 gene family. A Prosite pattern corresponding to the cell attachment sequence Arg-Gly-Asp was also found. The presence of this domain raised the intriguing possibility that APRG1B may be directly involved in membrane interactions and cell adhesion. We showed that the AP20 region was duplicated during mammalian evolution and homologous gene clusters were present in human chromosome 2 and syntenic mouse regions on chromosomes 1, 2, and 9. Interestingly, the HYA22 gene (human ortholog of the yeast YA22 gene) was located at the borders of both breakpoints, evolutionarily conserved gene cluster and homozygous deletions detected in lung, kidney and other cancers. NotI digestion revealed that the AP20 region was frequently and extensively methylated in renal carcinoma cell lines and tumor biopsies.

Discovery of frequent homozygous deletions in chromosome 3p21.3 LUCA and AP20 regions in renal, lung and breast carcinomas.

We searched for chromosome 3p homo- and hemizygous losses in 23 lung cancer cell lines, 53 renal cell and 22 breast carcinoma biopsies using 31 microsatellite markers located in frequently deleted 3p regions. In addition, two sequence-tagged site markers (NLJ-003 and NL3-001) located in the Alu-PCR clone 20 region (AP20) and lung cancer (LUCA) regions, respectively, were used for quantitative real-time PCR (QPCR). We found frequent (10-18%) homozygous deletions (HDs) in both 3p21.3 regions in the biopsies and lung cancer cell lines. In addition, we discovered that amplification of 3p is a very common (15-42.5%) event in these cancers and probably in other epithelial malignancies. QPCR showed that aberrations of either NLJ-003 or NL3-001 were detected in more than 90% of all studied cases. HDs were frequently detected simultaneously both in NLJ-003 or NL3-001 loci in the same tumour (P<3-10(-7)). This observation suggests that tumour suppressor genes (TSG) in these regions could have a synergistic effect. The exceptionally high frequency of chromosome aberrations in NLJ-003 and NL3-001 loci suggests that multiple TSG(s) involved in different malignancies are located very near to these markers. Precise mapping of 15 independent HDs in the LUCA region allowed us to establish the smallest HD region in 3p21.3C located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene). This region contains 17 genes. Mapping of 19 HDs in the AP20 region resulted in the localization of the minimal region to the interval flanked by D3S1298 and D3S3623 markers. Only four genes were discovered in this interval, namely, APRG1, ITGA9, HYA22 and VILL.

Increase of faecal tryptic activity relates to changes in the intestinal microbiome: analysis of Crohn's disease with a multidisciplinary platform.

OBJECTIVE: To investigate-by molecular, classical and functional methods-the microbiota in biopsies and faeces from patients with active Crohn's disease (CD) and controls. DESIGN: The microbiota in biopsies was investigated utilizing a novel molecular method and classical cultivation technology. Faecal samples were investigated by classical technology and four functional methods, reflecting alterations in short chain fatty acids pattern, conversion of cholesterol and bilirubin and inactivation of trypsin. RESULTS: By molecular methods we found more than 92% similarity in the microbiota on the biopsies from the two groups. However, 4.6% of microbes found in controls were lacking in CD patients. Furthermore, NotI representation libraries demonstrate two different clusters representing CD patients and controls, respectively. Utilizing conventional technology, Bacteroides (alt. Parabacteroides) was less frequently detected in the biopsies from CD patients than from controls. A similar reduction in the number of Bacteroides was found in faecal samples. Bacteroides is the only group of bacteria known to be able to inactivate pancreatic trypsin. Faecal tryptic activity was high in CD patients, and inversely correlated to the levels of Bacteroides. CONCLUSIONS: CD patients have compositional and functional alterations in their intestinal microbiota, in line with the global description hypothesis rather than the candidate microorganism theory. The most striking functional difference was high amount of faecal tryptic activity in CD patients, inversely correlated to the levels of Bacteroides in faeces.

Epigenetic analysis of childhood acute lymphoblastic leukemia.

We used a chromosome 3 wide NotI microarray for identification of epigenetically inactivated genes in childhood acute lymphoblastic leukemia (ALL). Three novel genes demonstrated frequent methylation in childhood ALL. PPP2R3A (protein phosphatase 2, regulatory subunit B", alpha) was frequently methylated in T (69%) and B (82%)-ALL. Whilst FBLN2 (fibulin 2) and THRB (thyroid hormone receptor, beta) showed frequent methylation in B-ALL (58%; 56% respectively), but were less frequently methylated in T-ALL (17% for both genes). Recently it was demonstrated that BNC1 (Basonuclin 1) and MSX1 (msh homeobox 1) were frequently methylated across common epithelial cancers. In our series of childhood ALL BNC1 was frequently methylated in both T (77%) and B-ALL (79%), whilst MSX1 showed T-ALL (25%) specific methylation. The methylation of the above five genes was cancer specific and expression of the genes could be restored in methylated leukemia cell lines treated with 5-aza-2'-deoxycytidine. This is the first report demonstrating frequent epigenetic inactivation of PPP2R3A, FBLN2, THRB, BNC1 and MSX1 in leukemia. The identification of frequently methylated genes showing cancer specific methylation will be useful in developing early cancer detection screens and for targeted epigenetic therapies.

High mutability of the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) in cancer.

BACKGROUND: Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas. METHODOLOGY/PRINCIPAL FINDINGS: We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1-2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3-5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines. CONCLUSIONS/SIGNIFICANCE: This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.

NotI subtraction and NotI-specific microarrays to detect copy number and methylation changes in whole genomes.

Methylation, deletions, and amplifications of cancer genes constitute important mechanisms in carcinogenesis. For genome-wide analysis of these changes, we propose the use of NotI clone microarrays and genomic subtraction, because NotI recognition sites are closely associated with CpG islands and genes. We show here that the CODE (Cloning Of DEleted sequences) genomic subtraction procedure can be adapted to NotI flanking sequences and to CpG islands. Because the sequence complexity of this procedure is greatly reduced, only two cycles of subtraction are required. A NotI-CODE procedure can be used to prepare NotI representations (NRs) containing 0.1-0.5% of the total DNA. The NRs contain, on average, 10-fold less repetitive sequences than the whole human genome and can be used as probes for hybridization to NotI microarrays. These microarrays, when probed with NRs, can simultaneously detect copy number changes and methylation. NotI microarrays offer a powerful tool with which to study carcinogenesis.

RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies.

Chromosome 3p21.3 region is frequently (>90%) deleted in lung and other major human carcinomas. We subdivided 3p21.3 into LUCA and AP20 subregions and discovered frequent homozygous deletions (10-18%) in both subregions. This finding strongly implies that they harbor multiple tumor suppressor genes involved in the origin and/or development of major epithelial cancers. In this study, we performed an initial analysis of RBSP3/HYA22, a candidate tumor suppressor genes located in the AP20 region. Two sequence splice variants of RBSP3/HYA22 (A and B) were identified, and we provide evidence for their tumor suppressor function. By sequence analysis RBSP3/HYA22 belongs to a gene family of small C-terminal domain phosphatases that may control the RNA polymerase II transcription machinery. Expression of the gene was drastically (>20-fold) decreased in 11 of 12 analyzed carcinoma cell lines and in three of eight tumor biopsies. We report missense and nonsense mutations in tumors where RBSP3/HYA22 was expressed, growth suppression with regulated transgenes in culture, suppression of tumor formation in severe combined immunodeficient mice, and dephosphorylation of ppRB by RBSP3/HYA22, presumably leading to a block of the cell cycle at the G1/S boundary.