D-glucuronyl C5-epimerase cell type specifically affects angiogenesis pathway in different prostate cancer cells.

D-glucuronyl C5-epimerase (GLCE) is involved in breast and lung carcinogenesis as a potential tumor suppressor gene, acting through inhibition of tumor angiogenesis and invasion/metastasis pathways. However, in prostate tumors, increased GLCE expression is associated with advanced disease, suggesting versatile effects of GLCE in different cancers. To investigate further the potential cancer-promoting effect of GLCE in prostate cancer, GLCE was ectopically re-expressed in morphologically different LNCaP and PC3 prostate cancer cells. Transcriptional profiles of normal PNT2 prostate cells, LNCaP, PC3 and DU145 prostate cancer cells, and GLCE-expressing LNCaP and PC3 cells were determined. Comparative analysis revealed the genes whose expression was changed in prostate cancer cells compared with normal PNT2 cells, and those differently expressed between the cancer cell lines (ACTA2, IL6, SERPINE1, TAGLN, SEMA3A, and CDH2). GLCE re-expression influenced mainly angiogenesis-involved genes (ANGPT1, SERPINE1, IGF1, PDGFB, TNF, IL8, TEK, IFNA1, and IFNB1) but in a cell type-specific manner (from basic deregulation of angiogenesis in LNCaP cells to significant activation in PC3 cells). Invasion/metastasis pathway was also affected (MMP1, MMP2, MMP9, S100A4, ITGA1, ITGB3, ERBB2, and FAS). The obtained results suggest activation of angiogenesis as a main molecular mechanism of pro-oncogenic effect of GLCE in prostate cancer. GLCE up-regulation plus expression pattern of a panel of six genes, discriminating morphologically different prostate cancer cell sub-types, is suggested as a potential marker of aggressive prostate cancer.

Tumor suppressor dual-specificity phosphatase 6 (DUSP6) impairs cell invasion and epithelial-mesenchymal transition (EMT)-associated phenotype.

Suppressive effects of DUSP6 in tumorigenesis and EMT-associated properties were observed. Dual-specificity phosphatase (DUSP6) is a MAP kinase phosphatase (MKP) negatively regulating the activity of ERK, one of the major molecular switches in the MAPK signaling cascade propagating the signaling responses during malignancies. The impact of DUSP6 in EMT and its contribution to tumor dissemination has not yet been characterized. Due to differences in tumor microenvironments affecting cell signaling during cancer progression, DUSP6 may play varying roles in tumor development. We sought to examine the potential role of DUSP6-mediated tumorigenesis and EMT-associated properties in two aerodigestive tract cancers, namely, esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Significant loss of DUSP6 was observed in 100% of 11 ESCC cell lines and 71% of seven NPC cell lines. DUSP6 expression was down-regulated in 40% of 30 ESCC tumor tissues and 75% of 20 NPC tumor tissues compared to their respective normal counterparts. Suppressive effects of DUSP6 in tumor formation and cancer cell mobility are seen in in vivo tumorigenicity assay and in vitro colony formation, three-dimensional Matrigel culture, cell migration and invasion chamber tests. Notably, overexpression of DUSP6 impairs EMT-associated properties. Furthermore, tissue microarray analysis reveals a clinical association of DUSP6 expression with better patient survival. Taken together, our study provides a novel insight into understanding the functional impact of DUSP6 in tumorigenesis and metastasis of ESCC and NPC.

Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma.

Chromosome 14 allelic loss is common in nasopharyngeal carcinoma (NPC) and may reflect essential tumor suppressor gene loss in tumorigenesis. An intact chromosome 14 was transferred to an NPC cell line using a microcell-mediated chromosome transfer approach. Microcell hybrids (MCHs) containing intact exogenously transferred chromosome 14 were tumor suppressive in athymic mice, demonstrating that intact chromosome 14 NPC MCHs are able to suppress tumor growth in mice. Comparative analysis of these MCHs and their derived tumor segregants identified 4 commonly eliminated tumor-suppressive CRs. Here we provide functional evidence that a gene, Mirror-Image POLydactyly 1 (MIPOL1), which maps within a single 14q13.1-13.3 CR and that hitherto has been reported to be associated only with a developmental disorder, specifically suppresses in vivo tumor formation. MIPOL1 gene expression is down-regulated in all NPC cell lines and in approximately 63% of NPC tumors via promoter hypermethylation and allelic loss. SLC25A21 and FOXA1, 2 neighboring genes mapping to this region, did not show this frequent down-regulated gene expression or promoter hypermethylation, precluding possible global methylation effects and providing further evidence that MIPOL1 plays a unique role in NPC. The protein localizes mainly to the nucleus. Re-expression of MIPOL1 in the stable transfectants induces cell cycle arrest. MIPOL1 tumor suppression is related to up-regulation of the p21(WAF1/CIP1) and p27(KIP1) protein pathways. This study provides compelling evidence that chromosome 14 harbors tumor suppressor genes associated with NPC and that a candidate gene, MIPOL1, is associated with tumor development.

Catalytic activity of Matrix metalloproteinase-19 is essential for tumor suppressor and anti-angiogenic activities in nasopharyngeal carcinoma.

The association of Matrix metalloproteinase-19 (MMP19) in the development of nasopharyngeal carcinoma (NPC) was identified from differential gene profiling, which showed MMP19 was one of the candidate genes down-regulated in the NPC cell lines. In this study, quantitative RT-PCR and Western blot analysis showed MMP19 was down-regulated in all seven NPC cell lines. By tissue microarray immunohistochemical staining, MMP19 appears down-regulated in 69.7% of primary NPC specimens. Allelic deletion and promoter hypermethylation contribute to MMP19 down-regulation. We also clearly demonstrate that the catalytic activity of MMP19 plays an important role in antitumor and antiangiogenesis activities in comparative studies of the wild-type and the catalytically inactive mutant MMP19. In the in vivo tumorigenicity assay, only the wild-type (WT), but not mutant, MMP19 transfectants suppress tumor formation in nude mice. In the in vitro colony formation assay, WT MMP19 dramatically reduces colony-forming ability of NPC cell lines, when compared to the inactive mutant. In the tube formation assay of human umbilical vein endothelial cells and human microvascular endothelial cells (HMEC-1), secreted WT MMP19, but not mutant MMP19, induces reduction of tube-forming ability in endothelial cells with decreased vascular endothelial growth factor (VEGF) in conditioned media detected by enzyme-linked immunosorbent assay (ELISA). The anti-angiogenic activity of WT MMP19 is correlated with suppression of tumor formation. These results now clearly show that catalytic activity of MMP19 is essential for its tumor suppressive and anti-angiogenic functions in NPC.

RBSP3 is frequently altered in premalignant cervical lesions: clinical and prognostic significance.

To understand the importance of frequent deletion of 3p22.3 in cervical carcinogenesis, alterations (deletion/methylation/expression) of the candidate genes STAC, MLH1, ITGA9, and RBSP3, located in the region, were analyzed in 24 cervical intraepithelial neoplasia (CIN) and 137 uterine cervical carcinoma (CACX) samples. In CIN, RBSP3 deletion (48%) and methylation (26%) were high compared with the other genes (4-9%). In CACX, alterations of these genes were as follows: deletion: STAC (54%) > MLH1 (46%) > RBSP3 (45%) > ITGA9 (41%), methylation: RBSP3 (25%) > ITGA9 (24%) > STAC (19%) > MLH1 (13%). Overall, alterations of RBSP3 showed association with CIN, whereas for STAC and MLH1, this frequency increased significantly from CIN --> Stage I/II and for ITGA9 from CIN --> Stage I/II and also from Stage I/II --> Stage III/IV. Quantitative mRNA expression analysis showed differential reduced expression of these genes in CACX concordant to their molecular alterations. The more active RBSP3B splice variant was underexpressed in CACX. RB1 was infrequently deleted in CACX. Concordance was seen between (i) inactivation of RBSP3 and intense p-RB1 nuclear immunostaining and (ii) low/absence of MLH1 expression and its molecular alterations in CACX. In normal cervical epithelium, p-RB1 immunostaining was low in differentiated cells, whereas MLH1 staining was seen in both nucleus and cytoplasm irrespective of differentiation stage. Alterations of the genes were significantly associated with poor prognosis. High parity (>or=5)/early sexual debut (

Functional characterization of THY1 as a tumor suppressor gene with antiinvasive activity in nasopharyngeal carcinoma.

THY1 was previously identified as a candidate tumor suppressor gene (TSG) associated with lymph node metastases in nasopharyngeal carcinoma (NPC) through functional studies. It was identified by oligonucleotide microarray analysis as an interesting differentially expressed gene. However, direct functional evidence is still lacking for THY1 being a TSG in NPC, as in vivo tumorigenicity assays have not been previously reported in our last study of THY1. In this study, a tetracycline-inducible expression vector, pETE-Bsd, was used to obtain stable transfectants of THY1. The stringent in vivo tumorigenicity assay results show that the activation of THY1 suppresses tumor formation of HONE1 cells in nude mice, and the tumor formation ability was restored in the presence of doxycycline (a tetracycline analog), when the gene is shut off. Functional inactivation of this gene is observed in all the tumors derived from the tumorigenic transfectant. The tumor suppressive effect could be repressed by knockdown of THY1 expression in nontumorigenic microcell hybrids. Further studies indicate that expression of THY1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)/G(1) phase. It greatly reduces the ability for anchorage-independent growth. The invasiveness of HONE1 cells was also inhibited by the expression of THY1. These findings suggest that THY1 is a TSG in NPC, which is involved in invasion and shows an association with tumor metastasis. Taken together, THY1 clearly plays an important functional role in tumor suppression in NPC.

Frequent alterations of the candidate genes hMLH1, ITGA9 and RBSP3 in early dysplastic lesions of head and neck: clinical and prognostic significance.

To understand the association between candidate tumor suppressor genes (TSGs) human mismatch repair protein homologue 1 (hMLH1), AP20 region gene 1 (APRG1), integrin alpha RLC (ITGA9), RB1 serine phosphates from human chromosome 3 (RBSP3) at chromosomal 3p22.3 region and development of head and neck squamous cell carcinoma (HNSCC), alterations (deletion/promoter methylation/expression) of these genes were analyzed in 65 dysplastic lesions and 84 HNSCC samples. Clinicopathological correlations were made with alterations of the genes. In HNSCC, deletion frequencies of hMLH1, ITGA9, and RBSP3 were comparatively higher than APRG1. Overall alterations (deletion/methylation) of hMLH1, ITGA9, and RBSP3 were high (45-55%) in mild dysplasia and comparable in subsequent stages of tumor progression. Quantitative RT-PCR analysis showed reduced expression of these genes in tumors concordant to their molecular alterations. An in vitro demethylation experiment by 5-aza-2'-deoxycytidine confirmed the promoter hypermethylation of RBSP3 in Hep2 and UPCI:SCC084 cell lines. Functionally less-active RBSP3A isoform was predominant in tumor tissues contrary to the adjacent normal tissue of tumors where more active RBSP3B isoform was prevalent. In immunohistochemical analysis, intense nuclear staining of hMLH1 and pRB (phosphorylated RB, the substrate of RBSP3) proteins were seen in the basal layer of normal epithelium. In tumors, concordance was seen between (i) low/intermediate level of hMLH1 expression and its molecular alterations; and (ii) intense nuclear staining of pRB and RBSP3 alterations. Poor patient outcome was seen with hMLH1 and RBSP3 alterations. Moreover, in absence of human papilloma virus (HPV) infection, tobacco-addicted patients with hMLH1, RBSP3 alterations, and nodal invasions showed poor prognosis. Thus our data suggests that dysregulation of hMLH1, ITGA9, and RBSP3 associated multiple cellular pathways are needed for the development of early dysplastic lesions of the head and neck.

Antiproliferative effect of D-glucuronyl C5-epimerase in human breast cancer cells.

BACKGROUND: D-glucuronyl C5-epimerase (GLCE) is one of the key enzymes in the biosynthesis of heparansulfate proteoglycans. Down-regulation of GLCE expression in human breast tumours suggests a possible involvement of the gene in carcinogenesis. In this study, an effect of GLCE ectopic expression on cell proliferation and viability of breast carcinoma cells MCF7 in vitro and its potential molecular mechanisms were investigated. RESULTS: D-glucuronyl C5-epimerase expression was significantly decreased in MCF7 cells compared to normal human breast tissue. Re-expression of GLCE inhibited proliferative activity of MCF7 cells according to CyQUANT NF Cell Proliferation Assay, while it did not affect their viability in Colony Formation Test. According to Cancer PathFinder RT Profiler PCR Array, antiproliferative effect of GLCE in vitro could be related to the enhanced expression of tumour suppressor genes р53 (+3.3 fold), E2F1 (+3.00 fold), BRCA1 (+3.5 fold), SYK (+8.1 fold) and apoptosis-related genes BCL2 (+4.2 fold) and NFKB1 (+2.6 fold). Also, GLCE re-expression in MCF7 cells considerably changed the expression of some genes involved in angiogenesis (IL8, +4.6 fold; IFNB1, +3.9 fold; TNF, +4.6 fold and TGFB1, -5.7 fold) and invasion/metastasis (SYK, +8.1 fold; NME1, +3.96 fold; S100A4, -4.6 fold). CONCLUSIONS: The ability of D-glucuronyl С5-epimerase to suppress proliferation of breast cancer cells MCF7 through the attenuated expression of different key genes involved in cell cycle regulation, angiogenesis and metastasis molecular pathways supports the idea on the involvement of the gene in regulation of breast cancer cell proliferation.

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).

Why do we not all die of cancer at an early age?

Traditionally, surveillance against cancer was thought of as mainly immunological. With the exception of tumors with a clear viral involvement, such as immunoblastomas (Epstein-Barr virus, EBV), cervical, anogenital, and skin carcinomas (HPV), and Kaposi's sarcoma (HHV-8) where the immune system is confronted with virally encoded, nonself targets, tumors with no viral involvement provide poor targets. Attempts to influence them by immunological means are akin to the breaking of tolerance. Robust nonimmunological surveillance mechanisms include DNA repair-based checkpoint functions, and the triggering of growth arrest and/or apoptosis pathways by DNA damage or by illegitimate oncogene activation (intracellular surveillance). There is emerging evidence for epigenetic surveillance, reflected in the stringency of imprinting. A fourth mechanism, intercellular surveillance, or microenvironmental control, is rapidly gaining momentum. It can be mediated by contactual controls or by differentiation-inducing signals. Somatic hybridization experiments have shown that tumorigenicity is usually suppressed in somatic hybrids between normal and malignant cells, as long as a fairly complete chromosome complement is maintained. Individual normal cell-derived chromosomes may have a similar suppressive effect. For example, genetic and molecular dissection of human 3p that shows frequent deletions in many human tumors has identified multiple tumor suppressor genes, which can inhibit both in vitro growth and in vivo tumorigenicity. In addition, five genes were found with an "asymmetric activity," capable of suppressing tumorigenicity, without affecting in vitro growth. These genes, LTF, L1MD1, HYAL1, HYAL2, and VHL, are of particular interest because they may be involved in microenvironmental control.

THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma.

Using oligonucleotide microarray analysis, THY1, mapping close to a previously defined 11q22-23 nasopharyngeal carcinoma (NPC) critical region was identified as showing consistent downregulated expression in the tumour segregants, as compared to their parental tumour-suppressing microcell hybrids (MCHs). Gene expression and protein analyses show that THY1 was not expressed in the NPC HONE1 recipient cells, tumour segregants, and other NPC cell lines; THY1 was exclusively expressed in the non-tumourigenic MCHs. The mechanism of THY1 gene inactivation in these cell lines was attributed to hypermethylation. Clinical study showed that in 65% of NPC specimens there was either downregulation or loss of THY1 gene expression. Using a tissue microarray and immunohistochemical staining, 44% of the NPC cases showed downregulated expression of THY1 and 9% lost THY1 expression. The frequency of THY1 downregulated expression in lymph node metastatic NPC was 63%, which was significantly higher than in the primary tumour (33%). After transfection of THY1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. These findings suggest that THY1 is a good candidate tumour suppressor gene in NPC, which is significantly associated with lymph node metastases.

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.

NotI flanking sequences: a tool for gene discovery and verification of the human genome.

A set of 22 551 unique human NotI flanking sequences (16.2 Mb) was generated. More than 40% of the set had regions with significant similarity to known proteins and expressed sequences. The data demonstrate that regions flanking NotI sites are less likely to form nucleosomes efficiently and resemble promoter regions. The draft human genome sequence contained 55.7% of the NotI flanking sequences, Celera's database contained matches to 57.2% of the clones and all public databases (including non-human and previously sequenced NotI flanks) matched 89.2% of the NotI flanking sequences (identity > or =90% over at least 50 bp, data from December 2001). The data suggest that the shotgun sequencing approach used to generate the draft human genome sequence resulted in a bias against cloning and sequencing of NotI flanks. A rough estimation (based primarily on chromosomes 21 and 22) is that the human genome contains 15 000-20 000 NotI sites, of which 6000-9000 are unmethylated in any particular cell. The results of the study suggest that the existing tools for computational determination of CpG islands fail to identify a significant fraction of functional CpG islands, and unmethylated DNA stretches with a high frequency of CpG dinucleotides can be found even in regions with low CG content.

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.

Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C.

Initial analysis identified the NPRL2/G21 gene located in 3p21.3C, the lung cancer region, as a strong candidate tumor suppressor gene. Here we provide additional evidence of the tumor suppressor function of NPRL2/G21. The gene has highly conserved homologs/orthologs ranging from yeast to humans. The yeast ortholog, NPR2, shows three highly conserved regions with 32 to 36% identity over the whole length. By sequence analysis, the main product of NPRL2/G21 encodes a soluble protein that has a bipartite nuclear localization signal, a protein-binding domain, similarity to the MutS core domain, and a newly identified nitrogen permease regulator 2 domain with unknown function. The gene is highly expressed in many tissues. We report inactivating mutations in a variety of tumors and cancer cell lines, growth suppression of tumor cells with tet-controlled NPRL2/G21 transgenes on plastic Petri dishes, and suppression of tumor formation in SCID mice. Screening of 7 renal, 5 lung, and 7 cervical carcinoma cell lines showed homozygous deletions in the 3' end of NPRL2 in 2 renal, 3 lung, and 1 cervical (HeLa) cell line. Deletions in the 3' part of NPRL2 could result in improper splicing, leading to the loss of the 1.8 kb functional NPRL2 mRNA. We speculate that the NPRL2/G21 nuclear protein may be involved in mismatch repair, cell cycle checkpoint signaling, and activation of apoptotic pathway(s). The yeast NPR2 was shown to be a target of cisplatin, suggesting that the human NPRL2/G21 may play a similar role. At least two homozygous deletions of NPRL2/G21 were detected in 6 tumor biopsies from various locations and with microsatellite instability. This study, together with previously obtained results, indicates that NPRL2 is a multiple tumor suppressor gene.

Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers.

Loss of heterozygosity (LOH) involving several chromosome 3p regions accompanied by chromosome 3p deletions are detected in almost 100% of small (SCLCs) and more than 90% of non-small (NSCLCs) cell lung cancers. In addition, these changes appear early in the pathogenesis of lung cancer and are found as clonal lesions in the smoking damaged respiratory epithelium including histologically normal epithelium as well as in epithelium showing histologic changes of preneoplasia. These 3p genetic alterations lead to the conclusion that the short arm of human chromosome 3 contains several tumor suppressor gene(s) (TSG(s)). Although the first data suggesting that 3p alterations were involved in lung carcinogenesis were published more than 10 years ago, only recently has significant progress been achieved in identifying the candidate TSGs and beginning to demonstrate their functional role in tumor pathogenesis. Some of the striking results of these findings has been the discovery of multiple 3p TSGs and the importance of tumor acquired promoter DNA methylation as an epigenetic mechanism for inactivating the expression of these genes in lung cancer. This progress, combined with the well known role of smoking as an environmental causative risk factor in lung cancer pathogenesis, is leading to the development of new diagnostic and therapeutic strategies which can be translated into the clinic to combat and prevent the lung cancer epidemic. It is clear now that genetic and epigenetic abnormalities of several genes residing in chromosome region 3p are important for the development of lung cancers but it is still obscure how many of them exist and which of the numerous candidate TSGs are the key players in lung cancer pathogenesis. We review herein our current knowledge and describe the most credible candidate genes.

Deletion mapping using quantitative real-time PCR identifies two distinct 3p21.3 regions affected in most cervical carcinomas.

We report chromosome 3p deletion mapping of 32 cervical carcinoma (CC) biopsies using 26 microsatellite markers located in frequently deleted 3p regions to detect loss of heterozygosity and homozygous loss. In addition, two STS markers (NLJ-003 and NL3-001) located in the 3p21.3 telomeric (3p21.3T) and 3p21.3 centromeric (3p21.3C) regions, respectively, were used for quantitative real-time PCR as TaqMan probes. We show that quantitative real-time PCR is reliable and sensitive and allows discriminating between 0, 1 and 2 marker copies per human genome. For the first time, frequent (five of 32 cases, i.e. 15.6%) homozygous deletions were demonstrated in CCs in both 3p21.3T and 3p21.3C regions. The smallest region homozygously deleted in 3p21.3C was located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene) and contains 17 genes previously defined as lung cancer candidate Tumor suppressor genes (TSG(s)). The smallest region homozygously deleted in 3p21.3T was flanked by D3S1298 and NL1-024 (D3S4285), excluding DLEC1 and MYD88 as candidate TSGs involved in cervical carcinogenesis. Overall, this region contains five potential candidates, namely GOLGA4, APRG1, ITGA9, HYA22 and VILL, which need to be analysed. The data showed that aberrations of either NLJ-003 or NL3-001 were detected in 29 cases (90.6%) and most likely have a synergistic effect (P<0.01). The study also demonstrated that aberrations in 3p21.3 were complex and in addition to deletions, may involve gene amplification as well. The results strongly suggest that 3p21.3T and 3p21.3C regions harbor genes involved in the origin and/or development of CCs and imply that those genes might be multiple TSG(s).

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.