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Multiple, bilateral renal carcinomas are a frequent occurrence in von Hippel-Lindau (VHL) disease. To elucidate the aetiological role of the VHL gene in human kidney tumorigenesis, localized and advanced tumours from 110 patients with sporadic renal carcinoma were analysed for VHL mutations and loss of heterozygosity (LOH). VHL mutations were identified in 57% of clear cell renal carcinomas analysed and LOH was observed in 98% of those samples. Moreover, VHL was mutated and lost in a renal tumour from a patient with familial renal carcinoma carrying the constitutional translocation, t(3;8)(p14;q24). The identification of VHL mutations in a majority of localized and advanced sporadic renal carcinomas and in a second form of hereditary renal carcinoma indicates that the VHL gene plays a critical part in the origin of this malignancy.

We describe a 3 generation family with members affected with papillary renal cell carcinoma, an uncommon histological type of renal cell carcinoma. Multiple tumors of varying size were present in both kidneys of affected family members. The disorder was not linked to polymorphic markers on chromosome 3p and there was no loss of heterozygosity at loci on 3p in renal tumors. The results suggest the presence of a renal cell carcinoma gene not located on 3p that predisposes to renal cell carcinoma with a distinct histological appearance. The inherited disorder in this family appears to be different from recognized hereditary cancer syndromes.

Chromosome 3 specific NotI microarrays containing 180 NotI linking clones associated with 188 genes were hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), lung (40 pairs) cervical (43 pairs), kidney (34 pairs of clear cell renal cell carcinoma), colon (24 pairs), ovarian (25 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. For the first time we showed that the gene LRRC3B was frequently methylated and/or deleted in breast carcinoma - 32% of samples, cervical - 35%, lung - 40%, renal - 35%, ovarian - 28%, colon - 33% and prostate cancer - 44%. To check these results bisulfite sequencing using cloned PCR products with representative two breast, one cervical, two renal, two ovarian and two colon cancer samples was performed. In all cases methylation was confirmed. Expression analysis using RT-qPCR showed that LRRC3B is strongly down-regulated at the latest stages of RCC and ovarian cancers. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. All these data suggest that LRRC3B gene could be involved in the process of carcinogenesis as a tumor suppressor gene.

Review on MST1R (macrophage stimulating 1 receptor), with data on DNA, on the protein encoded, and where the gene is implicated.

Von Hippel-Lindau disease (VHL) is an inherited multisystem neoplastic disorder. We prepared a 2.5-megabase (Mb) restriction map of the region surrounding the VHL gene and identified and characterized overlapping deletions in three unrelated patients affected with VHL. The smallest nested deletion (100 kb) was located within a 510-kb NruI fragment detected by 19-63'. The rearrangements detected will be useful in isolating and evaluating candidate cDNAs for the VHL gene. The detailed physical map will be useful in studying the organization and structure of genes in the VHL region.

As part of our effort to isolate and characterise the von Hippel-Lindau (VHL) disease gene, we constructed a physical map of chromosome 3p25-26 by fluorescence in situ hybridisation (FISH) studies on a panel of cytogenetic rearrangements involving this region. Biotinylated cosmid and lambda probes were hybridised to metaphase chromosome spreads and positioned with respect to each cytogenetic breakpoint. These studies unequivocally established the order of five loci linked to the VHL disease gene: cen-(RAF1,312)-D3S732-D3S1250-D3S601-D3S18 -pter and determined the position of three other probes within this map. These results ordered RAF1 and D3S732 for the first time, confirmed the localisation of D3S1250 between RAF1 and D3S601 and determined the position of D3S651 with respect to other chromosome 3p25-p26 loci. The establishment of an ordered set of cytogenetic aberrations will enable the rapid assignment of polymorphic and nonpolymorphic cloned sequences within the chromosome region 3p25-p26.

Hemizygous deletion of 3p25-pter is associated with a phenotype of profound growth failure, microcephaly, characteristic facial changes, and mental retardation. Since the severity may be quite variable, we have studied 3 cases of del 3p25-pter to define the clinical manifestations and the critical chromosome region for phenotypic expression. The patient we now report died at age 6 months and provided an opportunity for a detailed necropsy analysis for only the second time in a del(3p) patient. He had marked hypoplasia of all organs, hypomyelination of white matter, and multiple renal cortical microcysts. Ordered genomic markers from the distal regions of chromosome 3p aided in determining the parent of origin of each deletion and in defining the boundaries of the deleted chromosomal segments. The deleted markers distal to the RAF1 oncogene in 2 of the 3 patients were consistently hemizygous. One patient had an interstitial deletion based on evidence of diploid inheritance of one of the most distal loci (D3S17). Available genetic linkage maps suggest that the deletion spans at least 19 centimorgans (cM).

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 (≥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).

Transcriptional activation by hypoxia is mediated by the hypoxia-inducible factor (HIF) via binding to the hypoxia-responsive element (HRE). Hypoxia in solid tumors associates with poorer outcome of the disease and reliable cellular markers of tumor hypoxia would represent a valuable diagnostic marker and a potential therapeutic target. In this category, carbonic anhydrase IX (CAIX) is one of the most promising candidates. Here, we summarize the knowledge about transcriptional regulation of CA9. The HRE is the central regulatory element in the CA9 promoter, whereas other elements are limited to lesser roles of amplification of signals received at the HRE. The analysis of known mechanisms of activation of CA9 reveals the prominent role of the HIF-1 pathway. Experimental paradigms with uncoupled HIF-1alpha stability and transcriptional activity (pericellular hypoxia, proteasomal inhibitor) provide evidence that CA9 expression monitors transcriptional activity of HIF-1, rather than the abundance of HIF-1alpha. Furthermore, these paradigms could provide a corollary to some of the apparently discordant cases (CAIX+, HIF-1alpha-) or (CAIX-, HIF-1alpha+) observed in vivo. In conclusion, the existing data support the notion that CA9, due to the unique structure of its promoter, is one of the most sensitive endogenous sensors of HIF-1 activity.

Background Transmembrane CAIX and CAXII are members of the alpha carbonic anhydrase (CA) family. They play a crucial role in differentiation, proliferation, and pH regulation. Expression of CAIX and CAXII proteins in tumor tissues is primarily induced by hypoxia and this is particularly true for CAIX, which is regulated by the transcription factor, hypoxia inducible factor-1 (HIF-1). Their distributions in normal adult human tissues are restricted to highly specialized cells that are not always hypoxic. The human fetus exists in a relatively hypoxic environment. We examined expression of CAIX, CAXII and HIF-1α in the developing human fetus and postnatal tissues to determine whether expression of CAIX and CAXII is exclusively regulated by HIF-1. Results The co-localization of CAIX and HIF-1α was limited to certain cell types in embryonic and early fetal tissues. Those cells comprised the primitive mesenchyma or involved chondrogenesis and skin development. Transient CAIX expression was limited to immature tissues of mesodermal origin and the skin and ependymal cells. The only tissues that persistently expressed CAIX protein were coelomic epithelium (mesothelium) and its remnants, the epithelium of the stomach and biliary tree, glands and crypt cells of duodenum and small intestine, and the cells located at those sites previously identified as harboring adult stem cells in, for example, the skin and large intestine. In many instances co-localization of CAIX and HIF-1α was not evident. CAXII expression is restricted to cells involved in secretion and water absorption such as parietal cells of the stomach, acinar cells of the salivary glands and pancreas, epithelium of the large intestine, and renal tubules. Co-localization of CAXII with CAIX or HIF-1α was not observed. Conclusion The study has showed that: 1) HIF-1α and CAIX expression co- localized in many, but not all, of the embryonic and early fetal tissues; 2) There is no evidence of co-localization of CAIX and CAXII; 3) CAIX and CAXII expression is closely related to cell origin and secretory activity involving proton transport, respectively. The intriguing finding of rare CAIX-expressing cells in those sites corresponding to stem cell niches requires further investigation.

Genetic linkage studies were performed in 22 families with von Hippel-Lindau (VHL) disease by using polymorphic DNA markers from distal chromosome 3p. Linkage was detected between VHL disease and the markers D3S18 (Zmax = 6.6 at theta = 0.0, confidence interval (CI) 0.00-0.06), RAF1 (Zmax = 5.9 at theta = 0.06, CI 0.01-0.16), and THRB (Zmax 3.4 at theta = 0.11). Multipoint linkage analysis localized the VHL disease gene within a small region (approximately 8 cM) of 3p25-p26 between RAF1 and (D3S191, D3S225) and close to the D3S18 locus. There was no evidence of locus heterogeneity, and families with and without pheochromocytoma showed linkage to D3S18. The identification of DNA markers flanking the VHL disease gene allows reliable presymptomatic and prenatal diagnosis to be offered to informative families.

As part of an attempt to locate the von Hippel-Lindau locus (VHL) on chromosome 3, we evaluated 41 families with von Hippel-Lindau disease from the United States and Canada. One large family was identified whose disease phenotype was distinct from typical VHL. The most common disease manifestation was pheochromocytoma occurring in 57% (27/47) of affected family members. Few (4/47) affected family members had symptomatic spinal or cerebellar hemangioblastomas; no affected family member had renal cell carcinoma (0/47) or pancreatic cysts (0/24). Previously, genetic analysis demonstrated that the disease manifestations in this family were linked to RAF1 and D3S18, markers shown to be linked to typical VHL. These results suggest that there are mutant alleles at the VHL locus associated with distinct tissue specificities.

Background: We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions (e.g.G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo. Methodology/Principal Findings: The effect of expression HYAL1 and HYAL2 was studied by colony formation inhibition, growth curve and cell proliferation tests in vitro and tumour growth assay in vivo. Very modest growth inhibition was detected in vitro in U2020 lung and KRC/Y renal carcinoma cell lines. In the in vivo experiment stably transfected KRC/Y cells expressing HYAL1 or HYAL2 were inoculated into SCID mice (10 and 12 mice respectively). Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60–67%). Conclusions/Significance: The results suggest that the expression of either gene has led to inhibition of tumour growth in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment.

Transplanted donor lymphocytes infused during hematopoietic stem cell transplantation (HSCT) have been shown to cure patients with hematological malignancies. However, less is known about the effects of HSCT on metastatic solid tumors. Thus, a better understanding of the immune cells and their target antigens that mediate tumor regression is urgently needed to develop more effective HSCT approaches for solid tumors. Here we report regression of metastatic renal cell carcinoma (RCC) in patients following nonmyeloablative HSCT consistent with a graft-versus-tumor effect. We detected RCC-reactive donor-derived CD8(+) T cells in the blood of patients following nonmyeloablative HSCT. Using cDNA expression cloning, we identified a 10-mer peptide (CT-RCC-1) as a target antigen of RCC-specific CD8(+) T cells. The genes encoding this antigen were found to be derived from human endogenous retrovirus (HERV) type E and were expressed in RCC cell lines and fresh RCC tissue but not in normal kidney or other tissues. We believe this to be the first solid tumor antigen identified using allogeneic T cells from a patient undergoing HSCT. These data suggest that HERV-E is activated in RCC and that it encodes an overexpressed immunogenic antigen, therefore providing a potential target for cellular immunity.

We studied 25 families with von Hippel-Lindau disease (VHL) to locate VHL more precisely on chromosome 3. We found that VHL was linked to RAF1, confirming previous observations, and to two polymorphic DNA markers, D3S18 and D3S191. Multipoint linkage analysis indicated that the most likely location for VHL was in the interval between RAF1 and D3S18. D3S18 was located at 3p26. Genetic heterogeneity was not detected in this panel of von Hippel-Lindau disease families. The polymorphic markers RAF1, D3S18, and D3S191 should be useful in identifying asymptomatic gene carriers in VHL families and in guiding efforts at gene isolation.

Mutations in the von Hippel–Lindau tumour suppressor gene (VHL) cause the VHL hereditary cancer syndrome and occur in most sporadic clear cell renal cell cancers (CC-RCCs). The mechanisms by which VHL loss of function promotes tumour development in the kidney are not fully elucidated. Here, we analyse expression of PL6, one of the potential tumour suppressor genes from the critical 3p21.3 region involved in multiple common cancers. We classify PL6 as a Golgi-resident protein based on its perinuclear co-localization with GPP130 in all cells and tissues analysed. We show that PL6 RNA and protein expression is completely or partially lost in all analysed CC-RCCs and other VHL-deficient tumours studied, including the early precancerous lesions in VHL disease. The restoration of VHL function in vitro in the VHL-deficient CC-RCC cell lines was found to reinstate PL6 expression, thus establishing a direct link between VHL and PL6. Insensitivity of PL6 to hypoxia suggested that PL6 is regulated by VHL via a HIF-1-independent pathway. We ruled out mutations and promoter methylation as possible causes of PL6 down-regulation in CC-RCC. We hypothesize that loss of a putative PL6 secretory function due to VHL deficiency is an early and important event that may promote tumour initiation and growth. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Fus1 gene resides in the critical 3p21.3 human chromosomal region deleted in lung and breast cancers. Recently, the tumour suppressor properties of Fus1 were confirmed experimentally by intra-tumoural administration of Fus1 that suppressed experimental lung metastasis in mice. We generated Fus1-deficient mice that were viable, fertile, and demonstrated a complex immunological phenotype. Animals with a disrupted Fus1 gene developed signs of autoimmune disease, such as vasculitis, glomerulonephritis, anaemia, circulating autoantibodies, and showed an increased frequency of spontaneous vascular tumours. Preliminary analysis of immune cell populations revealed a consistent defect in NK cell maturation in Fus1 null mice that correlated with changes in the expression of IL-15. Injection of IL-15 into Fus1 knockout mice completely rescued the NK cell maturation defect. Based on these results, we propose the hypothesis that Fus1 deficiency affects NK cell maturation through the reduction of IL-15 production but does not directly alter their developmental capacity. Since acquired immunity was not affected in Fus1-deficient animals, we suggest a relationship between the Fus1 protein and the regulation of innate immunity via IL-15 production. The increased frequency of spontaneous cancers and the development of an autoimmune syndrome in Fus1 null mice imply that these mice could serve as a model for studying molecular mechanisms of anti-tumour immunity and autoimmunity.

Chromosome 3p plays an important role in tumorigenesis in many cancers, including nasopharyngeal carcinoma (NPC). We have previously shown chromosome 3p can suppress tumor growth in vivo by using the monochromosome transfer approach, which indicated the chromosome 3p21.3 region was critical for tumor suppression. BLU/ZMYND10 is one of the candidate tumor suppressor genes mapping in the 3p21.3 critical region and is a candidate TSG for NPC. By quantitative RT-PCR, it is frequently downregulated in NPC cell lines (83%) and NPC biopsies (80%). However, no functional studies have yet verified the functional role of BLU/ZMYND10 as a tumor suppressor gene. In the current study, a gene inactivation test (GIT) utilizing a tetracycline regulation system was used to study the functional role of BLU/ZMYND10. When BLU/ZMYND10 is expressed in the absence of doxycycline, the stable transfectants were able to induce tumor suppression in nude mice. In contrast, downregulation of BLU/ZMYND10 in these tumor suppressive clones by doxycycline treatment restored the tumor formation ability. This study provides the first significant evidence to demonstrate BLU/ZMYND10 can functionally suppress tumor formation in vivo and is, therefore, likely to be one of the candidate tumor suppressor genes involved in NPC. Published 2006 Wiley-Liss, Inc.

Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung and breast cancer cell lines U2020, NCI H2198, and HCC38. It is DUTTI (Deleted in U Twenty Twenty), also known as ROBO1, FLJ21882, and SAX3, according to HUGO. DUTTI, the human ortholog of the fly gene ROBO, has homology with NCAM proteins. Extensive analyses of DUTTI in lung cancer have not revealed any mutations, suggesting that another gene(s) at this location could be of importance in lung cancer initiation and progression. Here, we report the discovery of a new, small, homozygous deletion in the small cell lung cancer (SCLC) cell line GLC20, nested in the overlapping, critical region. The deletion was delineated using several polymorphic markers and three overlapping PI phage clones. Fiber-FISH experiments revealed the deletion was approximately 130 kb. Comparative genomic sequence analysis uncovered short sequence elements highly conserved among mammalian genomes and the chicken genome. The discovery of two EST clusters within the deleted region led to the isolation of two noncoding RNA (ncRNA) genes. These were subsequently found differentially expressed in various tumors when compared to their normal tissues. The ncRNA and other highly conserved sequence elements in the deleted region may represent miRNA targets of importance in cancer initiation or progression. Published 2006 Wiley-Liss, Inc.(dagger).

Background: STRA13 is a bHLH transcription factor that plays a crucial role in cell differentiation, proliferation, apoptosis, and response to hypoxia. Objective: To assess STRA13 involvement in carcinogenesis and evaluate its diagnostic value. Methods: A comprehensive analysis was undertaken of the endogenous protein expression in 389 normal and corresponding malignant specimens, using newly generated polyclonal antibodies. Results: STRA13 was commonly expressed in epithelial cells of normal and neoplastic tissues where it was confined mostly to the nucleus. Intense cytoplasmic STRA13 immunoreactivity was characteristic of myoepithelial and differentiated squamous epithelial cells of all organ sites and their neoplastic counterparts, suggesting application of STRA13 as a myoepithelial cell marker. A distinctive apical granular cytoplasmic staining pattern observed in the pancreas and large intestine was retained in corresponding metastatic carcinomas, providing for identification of the primary sites of these disseminating tumours. In less differentiated tumours there was a tendency to lose the cytoplasmic staining or to switch to nuclear STRA13 staining. Analysis of STRA13, HIF-1α, and CAIX expression patterns in a large set of various tumours substantiated the association of STRA13 with HIF-1α expression and hypoxia in vivo. Investigation of the molecular mechanisms of STRA13 nucleo-cytoplasmic shuttling suggested that STRA13 employs nuclear import/export that utilises the NLS/NES motifs situated within the N-terminus and in the middle of the protein. Conclusions: STRA13 may serve as a marker for myoepithelial cells, for the degree of tumour differentiation, and for identification of the primary site of certain metastatic tumours. In combination with CAIX and CAXII markers, it may lead to a more accurate classification of all renal carcinomas.

The expression of six chromosome 3p21.3 candidate tumor suppressor genes (BLU, FUS2, HYAL2, NPRL2, RASSF1A, and SEMA3B) in esophageal squamous cell carcinoma (ESCC) has been investigated. Reduced expression of BLU was detected in some ESCC cell lines and tumor tissues and the difference was quantitated by real-time quantitative polymerase chain reaction. Methylation specific-PCR revealed the down-regulation of BLU by epigenetic inactivation. However, exogenous expression of BLU did not functionally suppress tumorigenicity in nude mice. These results suggest that over-expression of BLU alone is not sufficient to inhibit tumorigenicity. Further studies on BLU interacting proteins are required to elucidate the possible role of BLU in the development of ESCC.

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.

CACNA2D2 is a putative tumor suppressor gene located in the human chromosome 3p21.3 region that shows frequent allelic imbalances in lung, breast, and other cancers. The alpha2delta-2 protein encoded by the gene is a regulatory subunit of voltage-dependent calcium channels and is expressed in brain, heart, and other tissues. Here we report that mice homozygous for targeted disruption of the Cacna2d2 gene exhibit growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells followed by Purkinje cell depletion, enhanced susceptibility to seizures, and cardiac abnormalities. The Cacna2d2(tm1NCIF) null phenotype has much in common with that of Cacna1a mutants, such as cerebellar neuro-degeneration associated with ataxia, seizures, and premature death. A tendency to bradycardia and limited response of null mutants to isoflurane implicate alpha2delta-2 in sympathetic regulation of cardiac function. In summary, our findings provide genetic evidence that the alpha2delta-2 subunit serves in vivo as a component of P/Q-type calcium channels, is indispensable for the central nervous system function, and may be involved in hereditary cerebellar ataxias and epileptic disorders in humans.

Immune responses to invading pathogens are mediated largely through a family of transmembrane Toll-like receptors and modulated by a number of downstream effectors. In particular, a family of four interleukin 1 receptor-associated kinases (IRAK) regulates responsiveness to bacterial endotoxins. Pharmacological targeting of particular IRAK components may be beneficial for treatment of bacterial infections. Here, we studied transcriptional regulation of the human IRAK2 gene. Analysis of the IRAK2 promoter region reveals putative binding sites for several transcriptional factors, including ZIP (EGR1 and SP1), CTCF and AP-2beta. Deletion of the ZIP or AP-2 sites did not significantly affect IRAK2 promoter activity in naive and endotoxin-treated mononuclear cells, in dormant and activated Jurkat T-cells, in lung and kidney cells. In contrast, we found that CTCF plays a major role in IRAK2 transcription. An electrophoretic mobility shift assay of the DNA fragments containing the IRAK2 CpG island, revealed a single high-affinity binding site for the transcriptional regulator and a chromatin insulator protein, CTCF. This assay revealed a CTCF-binding site within the mouse Irak2 promoter. The presence of the CTCF protein in human IRAK2 promoter was confirmed by chromatin immunoprecipitation assay. Specific residues that interacted with the CTCF protein, were identified by methylation interference assay. In all cell lines analyzed, including cells of lung, renal, monocytic and T-cell origin, the IRAK2 luciferase reporter construct, containing an intact CTCF-binding site, showed strong promoter activity. However, IRAK2 promoter activity was decreased dramatically for the constructs with a mutated CTCF-binding site.

RASSF1A, a major member of the RASSF1 gene family, is silenced by promoter methylation at a high frequency in a large number of human solid tumors. Controlled expression of RASSF1A reverts the tumorigenic phenotype of several human cancer cell lines. Here we investigated another main isoform, RASSF1C, and compared it with RASSF1A in the gene inactivation test (GIT), based on a tetracycline regulation system. In the small-cell lung cancer (SCLC) line U2020, only RASSF1A has shown growth inhibitory activity in vitro, while in the prostate cell line LNCaP and renal cell carcinoma (RCC) line KRC/Y both RASSF1A and RASSF1C showed similar (approximately 90%) suppressing activity in vitro. Both RASSF1C and RASSF1A suppressed the tumorigenicity of the KRC/Y RCC cell line in SCID mice. Mutations, deletions and loss of expression of RASSF1A and RASSF1C transgenes were identified in all 15 grown SCID tumors. In contrast, the mutant RASSF1A containing Cys65Arg and Val211Ala had reduced growth suppression activity both in vitro and in vivo and did not show any further changes in four grown SCID tumors. In addition, RASSF1C was shown to induce cell cycle arrest in KRC/Y cells. These results strongly imply that like RASSF1A the RASSF1C gene could serve a tumor suppressor function.

The FUS2 gene, encoding a novel cytoplasmic acetyltransferase, resides in the tumor suppressor gene region on human chromosome 3p21.3 and is considered a promising candidate tumor suppressor gene. We have identified a new single nucleotide polymorphism (SNP), c767A/T, in the coding region of the gene. The polymorphism leads to a non-conservative amino acid change (R222W) located between the acetyltransferase (GNAT) and the proline-rich domains of the protein. We have analyzed 254 subjects included in 14 sub-populations. The occurrence of the SNP varies with the ethnicity of the population, suggesting that this SNP could be a valuable biomarker for population genetics. It is most prevalent in various Asian populations (T allele frequency>0.54), followed by the Canadian polar Inuit (T allele frequency=0.3), African American (T allele frequency=0.17), and Caucasian population (T allele frequency=0.1). Since nasopharyngeal carcinoma (NPC) is frequent in Southern China, Taiwan, Borneo and polar Canada, we further tested for the possible association of the FUS2 SNP with this form of endemic cancer. Our analysis, albeit limited, suggests no likely association between NPC and the FUS2 gene polymorphism. Further large-scale case-control studies are necessary and warranted to prove the strength of this contention.

STRA13 is a pVHL-dependent bHLH transcription factor up-regulated on the mRNA level in multiple cancer cell lines and implicated recently in the regulation of immune cell homeostasis and autoimmunity. In searching for STRA13-interacting proteins with oncogenic potential by the yeast two-hybrid screening, we identified STAT3 beta as a STRA13-binding partner. We showed that STRA13 binds predominantly to phosphorylated (active) STAT3 alpha and beta isoforms via its HLH and C-terminal regions. We also found that STRA13 was able to activate transcription from STAT-dependent cis-elements. Expression of endogenous STRA13 was shown to be cytokine-inducible, consistent with STRA13 involvement in STAT-dependent transcription regulation. We demonstrated that the STAT3-regulated promoter of the pro-apoptotic Fas gene was activated upon STRA13 over-expression and that co-expression of STRA13 with STAT3 beta or STAT3 alpha modulated the transcriptional outcome. Forced expression of STRA13 induced apoptosis, in agreement with the STRA13 activation effect on the Fas promoter. Simultaneous expression of STRA13 and STAT3 beta resulted in alleviation of the STRA13 pro-apoptotic effect. Thus, for the first time, we identify STRA13 as a STAT3 partner and provide a consistent line of evidence for STRA13 involvement into regulation of apoptosis via the STAT pathways.

RON is a receptor tyrosine kinase of the MET family that is involved in cell proliferation, cell survival, and cell motility in both normal and disease states. Macrophage-stimulating protein (MSP) is the RON ligand whose binding to RON causes receptor activation. RON is a trans-membrane heterodimer comprised of one and one -chain originating from a single-chain precursor and held together by several disulfide bonds. The intracellular part of RON contains the kinase domain and regulatory elements. The extracellular region is characterized by the presence of a sema domain (a stretch of 500 amino acids with several highly conserved cysteine residues), a PSI (plexin, semaphorins, integrins) domain, and four immunoglobulin-like folds. Here we show that a soluble, secreted molecule representing the sema domain of RON (referred to as ronsema) has a dominant negative effect on the ligandinduced receptor activation and is capable of inhibiting RON-dependent signaling pathways and cellular responses. Results suggest that the sema domain of RON participates in ligand binding by the full-length receptor. The ability of ron-sema to suppress growth of MSPresponsive cells in culture, including cancer cells, points to a potential therapeutic use of this molecule, and forced expression of it could potentially be used as a gene therapy tool for treating MSP-dependent types of cancer. The human receptor tyrosine kinases MET and RON (MST1R) (and their respective orthologs in other species) form a unique, two-member gene family that encodes proteins with identical modular structure that may perform similar functions. Oncogenic activating mutations of MET were discovered in hereditary renal carcinoma of the papillary type (1, 2) and a variety of common cancers (3). Oncogenic amplification and overexpression of MET were also reported previously (3). The involvement of RON in carcinogenesis is much less understood. We have recently discovered that RON is negatively controlled by the tumor suppressor protein HYAL2 and becomes activated in a ligand-independent mechanism upon HYAL2 removal, leading to cell transformation (4). An oncogenic function of

RASSF1A, a recently identified candidate tumor suppressor gene, was found to be inactivated in lung cancer and other tumor types. We sought to understand the role of RASSF1A in head and neck cancer.We analyzed the status of RASSF1A and presence of high-risk human papilloma virus (HPV) in head and neck cancer squamous cell carcinoma (HNSCC) cell lines and primary tumors. We used methylation-specific PCR to detect promoter hypermethylation and direct sequence analysis to detect point mutations in primary tumors and cell lines. 5-aza-2-deoxycytidine was used to demethylate the RASSF1A promoter in cell lines.Promoter methylation of RASSF1A was detected in 42.9% (3 of 7) cell lines and 15% (7 of 46) primary tumors but not in the normal control DNA. Direct sequence analysis revealed a point mutation in a cell line and another in a primary HNSCC. After treatment with 5-aza-2-deoxycytidine, re-expression and demethylation of RASSF1A gene were detected in cell lines with promoter hypermethylation. HPV DNA was detected in 34.7% (16 of 46) primary HNSCC. We found a significant inverse correlation between RASSF1A promoter methylation and HPV infection (P = 0.038).Our results suggest that RASSF1A is inactivated in a subset of HNSCC primary tumors. Moreover, an inverse correlation between RASSF1A and HPV supports a biological mechanism in which both RASSF1A promoter methylation and HPV infection abrogate the same pathway in tumorigenesis.

Recently, we have identified a new putative tumor suppressor gene, RASSF1A (Ras association domain family 1A gene), located at human chromosome 3p21.3, the segment that is often lost in many types of human cancers. The RASSF1A promoter was shown to be frequently hypermethylated in various epithelial tumors, including small cell lung, breast, bladder, prostate, gastric, and renal cell carcinomas. In this study, we have analyzed the methylation status of the RASSF1A gene in primary human cervical cancers and in eight cervical cancer cell lines. The RASSF1A promoter is hypermethylated in 4 of 42 (= 10%) of squamous cell carcinomas, in 4 of 19 (= 21%) of adenosquamous carcinomas, and in 8 of 34 (= 24%) of cervical adenocarcinomas. Although in adenocarcinomas, methylation of RASSF1A and presence of human papillomavirus (HPV) type 16 or 18 sometimes coexisted, not a single case of HPV-16/18-positive squamous cell carcinomas had RASSF1A methylation. Similarly, in all eight analyzed cervical cell lines, RASSF1A inactivation and HPV infection were mutually exclusive (Fisher's exact test; P = 0.0357): two HPV-negative cervical cancer cell lines had a methylated and silenced RASSF1A promoter (C-33A and HT-3), whereas the other six HPV-positive lines expressed RASSF1A mRNA (ME 180, MS751, SiHa, C-4I, HeLa, and CaSki). For cervical tumors and cell lines combined, the Pearson's chi(2) test (chi(2) = 3.99; Por= 0.05) indicates a borderline-significant reverse correlation between inactivation of RASSF1A and the presence of high-risk HPVs. Our data imply that the presence of HPVs in cervical carcinomas alleviates the requirement for RASSF1A inactivation and suggests that these two events may engage the same tumorigenic pathway.

Metastatic renal cell carcinoma (RCC) has a poor prognosis and an unpredictable course. To date, there are no molecular markers which can reliably predict RCC outcome. We investigated whether a novel kidney cancer marker, carbonic anhydrase IX (CAIX), is associated with progression and survival.Immunohistochemical analysis using a CAIX monoclonal antibody was performed on tissue microarrays constructed from paraffin-embedded specimens from patients (N = 321) treated by nephrectomy for clear cell RCC. CAIX staining was correlated with response to treatment, clinical factors, pathologic features, and survival.CAIX staining was present in 94% of clear cell RCCs. Survival tree analysis determined that a cutoff of 85% CAIX staining provided the most accurate prediction of survival. Low CAIX (/=85%) staining was an independent poor prognostic factor for survival for patients with metastatic RCC, with a hazard ratio of 3.10 (P0.001). CAIX significantly substratified patients with metastatic disease when analyzed by T stage, Fuhrman grade, nodal involvement, and performance status (P0.001, = 0.001, = 0.009, = 0.005, respectively). For patients with nonmetastatic RCC and at high risk for progression, low CAIX predicted a worse outcome similar to patients with metastatic disease (P = 0.058). Overall expression of CAIX decreased with development of metastasis; as demonstrated by the lower CAIX staining levels in metastatic lesions relative to matched primary tumor specimens (P = 0.036).On the basis of our data, CAIX is the most significant molecular marker described in kidney cancer to date. Decreased CAIX levels are independently associated with poor survival in advanced RCC. CAIX reflects significant changes in tumor biology, which should be used to predict clinical outcome and identify high-risk patients in need for adjuvant immunotherapy and CAIX-targeted therapies.

Transcription of the gene coding for the tumor-associated antigen MN/carbonic anhydrase IX (CAIX) is regulated by hypoxia-inducible factor 1 (HIF-1). Previous studies identified CAIX expression in areas adjacent to hypoxic regions in solid tumors and suggested supplementary/alternative modes of regulation. To better understand the mechanisms activating CAIX expression, we characterized the cell density-dependent induction of CAIX in HeLa cells. This process is anchorage and serum independent and is not mediated by a soluble factor, decreased pH, or lowered glucose concentration. Stabilization of HIF-1 alpha was not observed in dense cultures. In contrast to sparse cell culture conditions, phosphatidylinositol 3'-kinase (PI3K) activity was significantly increased in dense HeLa cultures. The PI3K inhibitors LY294002 and wortmannin inhibited CAIX expression in dense cultures in a dose-dependent manner, specifically targeting the CA9 promoter (-173/+31 region) that was transactivated by constitutively active p110 PI3K subunit. The mechanism controlling CAIX expression in dense cultures is, however, dependent on lowered O(2) tension because stirring abrogates induction of CAIX expression. Hypoxia- and cell density-induced CAIX expressions were mediated by two seemingly independent mechanisms, as documented by the additive effect of increased cell density and treatment with the hypoxia-mimic CoCl(2) on levels of CAIX expression. The minimal cell density-dependent region within the CA9 promoter consists of the juxtaposed protected region 1 and hypoxia-response elements. However cell density-dependent CAIX expression was abrogated in the HIF-1 alpha-deficient Kal3.5 cells, suggesting an important role of HIF-1 in the corresponding mechanism. Thus, induction of CAIX in high-density cultures requires separate but interdependent pathways of PI3K activation and a minimal level of HIF-1 alpha. These interdependent pathways function at a lowered O(2) concentration that is, however, above that necessary for HIF-1 alpha stabilization.

Methylation, deletions, and amplifications of cancer genes constitute important mechanisms in carcinogenesis. For genome-wide analysis of these changes, we propose the use of Not I clone microarrays and genomic subtraction, because Not I 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 Not I flanking sequences and to CpG islands. Because the sequence complexity of this procedure is greatly reduced, only two cycles of subtraction are required. A Not I-CODE procedure can be used to prepare Not I 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 Not I microarrays. These microarrays, when probed with NRs, can simultaneously detect copy number changes and methylation. Not I microarrays offer a powerful tool with which to study carcinogenesis.

To ascertain the involvement of human chromosome 3p and its established critical TSG regions in various epithelial malignancies, 21 polymorphic and 2 nonpolymorphic 3p markers were allelotyped in nonpapillary RCC, NSCLC, CC and BC from a total of 184 patients. LOH was observed with high frequency in all types of cancer studied: RCC (52/57, 91%), BC (41/51, 80%), NSCLC (30/40, 75%) and CC (27/36, 75%). Interstitial deletions, believed to signal TSG inactivation, were verified using the “L-allele rule” and real-time quantitative PCR. Significant correlation was observed between DNA copy numbers for 2 nonpolymorphic STS markers and LOH data for adjacent polymorphic loci. Interstitial deletions in 3p were demonstrated for all cancer types studied. However, the distribution of different types of deletion was characteristic for tumors from various locations. Large terminal deletions were predominantly seen in RCC and NSCLC (51% and 40%, respectively), correlating with clear cell RCC and squamous cell carcinomas of the lung. In addition to the LUCA region at 3p21.3 (centromeric), we found that the AP20 region (3p21.3, telomeric) was frequently affected in all 4 cancers, suggesting that this newly defined critical region contains multiple TSGs. Moreover, at least 3 candidate cancer-specific loci were identified. The telomeric 3p26.1-p25.3 region was predominantly deleted in RCC and NSCLC. The D3S1286 and D3S3047 markers (3p25.2-p24.3) were deleted nonrandomly in NSCLC. High-frequency LOH was detected in a segment mapped closely distal to the LUCA site (3p21.3), around the D3S2409 and D3S2456 markers. © 2002 Wiley-Liss, Inc.

The VHL gatekeeper tumour suppressor gene is inactivated in the familial cancer syndrome von Hippel-Lindau disease and in most sporadic clear cell renal cell carcinomas. Recently the VHL gene product has been identified as a specific component of a SCF-like complex, which regulates proteolytic degradation of the hypoxia inducible transcription factors HIF-1 and HIF-2. pVHL is critical for normal development and mRNA expression studies suggest a role in nephrogenesis. Despite the importance of VHL in oncogenesis and development, little is known about the regulation of VHL expression. To investigate VHL promoter activity, we performed comparative sequence analysis of human, primate, and rodent 5' VHL sequences. We then proceeded to deletion analysis of regions showing significant evolutionary conservation between human and rat promoter sequences, and defined two positive and one negative regulatory regions. Analysis of specific putative transcription factor binding sites identified a functional Sp1 site, which was shown to be a regulatory element. Overlapping Sp1/AP2 sites were also identified and candidate E2F1 binding sites evaluated. Three binding sites for as yet unidentified transcription factors were mapped also. These investigations provide a basis for elucidating the regulation of VHL expression in development, the molecular pathology of epigenetic silencing of VHL in tumourigenesis, and suggest a possible link between Sp1, VHL, and nephrogenesis.

We analyzed expression status of the recently identified tumor suppressor geneRASSF1A in primary prostate carcinomas and in prostate cell lines. We found complete methylation of the RASSF1A promoter in 63% of primary microdissected prostate carcinomas (7 of 11 samples). The remaining 4 samples (37%) were partially methylated, possibly because of contamination with normal cells. No promoter methylation was observed in matching normal prostate tissues. High levels of RASSF1A transcript and no methylation of RASSF1A promoter were found in explanted primary normal prostate epithelial and stromal cells. Complete silencing and methylation of RASSF1A promoter was observed in five widely used prostate carcinoma cell lines, which acquired the ability to grow in culture spontaneously, including LNCaP, PC-3, ND-1, DU-145, 22Rv1, and one primary prostate carcinoma immortalized by overexpression of the human telomerase catalytic subunit (RC-58T/hTERT). However, no silencing of RASSF1A was found in four other prostate carcinoma cell lines, which were adapted for cell culture after transformation with human papillomaviral DNA. Suppression of cell growth in vitro was demonstrated after the reintroduction of RASSF1A-expressing construct into LNCaP prostate carcinoma cells. Our data implicate the RASSF1A gene in human prostate tumorigenesis.

Enzootic nasal tumor virus (ENTV) induces nasal epithelial cancer in infected sheep, but it is a simple retrovirus lacking a known oncogene. ENTV is closely related to jaagsiekte sheep retrovirus (JSRV), which also causes cancer in sheep but in the epithelial cells of the lower airways and alveoli. Here we show that as with JSRV, the envelope (Env) protein of ENTV can transform cultured cells and thus is likely to be responsible for oncogenesis in animals. In addition, the ENTV Env protein mediates virus entry using the same receptor as does JSRV Env, the candidate tumor suppressor Hyal2. However, ENTV Env mediates entry into cells from a more restricted range of species than does JSRV, and based on this finding we have identified amino acid regions in the Env proteins that are important for virus entry. Also, because ENTV does not efficiently use human Hyal2 as a receptor, we cloned the ovine Hyal2 cDNA and show that the encoded protein functions as an efficient receptor for both ENTV and JSRV. In summary, although ENTV and JSRV use the same cell surface receptor for cell entry and apparently transform cells by the same mechanism, they induce cancer in different tissues of infected sheep, indicating that oncogenesis is regulated at some other level. The transcriptional regulatory elements in these viruses are quite different, indicating that tissue-specific oncogenesis is likely regulated at the level of viral gene expression.

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope ( env ) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for ≈25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Recently we identified FUS1 as a candidate tumor suppressor gene (TSG) in the 120 kb 3p21.3 critical region contained in nested lung and breast cancer homozygous deletions. Mutation of FUS1 is infrequent in lung cancers which we have confirmed in 40 other primary lung cancers. In addition, we found no evidence for FUS1 promoter region methylation. Because haploinsufficiency or low expression of Fus1 may play a role in lung tumorigenesis, we tested the effect of exogenously induced overexpression of Fus1 protein and found 60–80% inhibition of colony formation for non-small cell lung cancer lines NCI-H1299 (showing allele loss for FUS1) and NCI-H322 (containing only a mutated FUS1 allele) in vitro. By contrast, a similar level of expression of a tumor-acquired mutant form of FUS1 protein did not significantly suppress colony formation. Also, induced expression of Fus1 under the control of an Ecdysone regulated promoter decreased colony formation 75%, increased the doubling time twofold, and arrested H1299 cells in G1. In conclusion, our data are consistent with the hypothesis that FUS1 may function as a 3p21.3 TSG, warranting further studies of its function in the pathogenesis of human cancers.

Debora Angeloni,∗ Joshua D. Lee, Bruce E. Johnson, Bin Tean Teh, Michael Dean, Michael I. Lerman and Esta Sterneck Laboratory of Immunobiology, National Cancer Institute at Frederick, Frederick, MD 21702, USA, Dana-Farber Cancer Institute, Boston, MA 02115, USA, Van Andel Research Institute, Gran Rapids, MI 49503, USA, Laboratory of Genomic Diversity, National Cancer Institute at Frederick, Frederick, MD 21702, USA and Regulation of Cell Growth Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA

The candidate tumour suppressor gene, LUCA-15, maps to the lung cancer tumour suppressor locus 3p21.3. Overexpression of an alternative RNA splice variant of LUCA-15 has been shown to retard human Jurkat T cell proliferation and to accelerate CD95-mediated apoptosis. An antisense cDNA to the 3'-UTR of this splice variant was able to suppress CD95-mediated apoptosis. Here, we report that overexpression of LUCA-15 itself suppresses CD95-mediated apoptosis in Jurkat cells. This suppression occurs prior to the final execution stage of the CD95 signalling pathway, and is associated with up-regulation of the apoptosis inhibitory protein Bcl-2. LUCA-15 overexpression is also able to inhibit apoptosis induced by the protein kinase inhibitor staurosporine, but is not able to significantly suppress apoptosis mediated by the topoisomerase II inhibitor etoposide. These findings suggest that LUCA-15 is a selective inhibitor of cell death, and confirm the importance of the LUCA-15 genetic locus in the control of apoptosis.

Previously we analysed overlapping homozygous deletions in lung and breast tumours/tumour lines and defined a small region of 120 kb (part of LCTSGR1) at 3p21.3 that contained putative lung and breast cancer tumour suppressor gene(s) (TSG). Eight genes including RASSF1 were isolated from the minimal region. However, extensive mutation analysis in lung tumours and tumour lines revealed only rare inactivating mutations. Recently, de novo methylation at a CpG island associated with isoform A of RASSF1 (RASSF1A) was reported in lung tumours and tumour lines. To investigate RASSF1A as a candidate TSG for various cancers, we investigated: (a) RASSF1A methylation status in a large series of primary tumour and tumour lines; (b) chromosome 3p allele loss in lung tumours and (c) RASSF1 mutation analysis in breast tumours. RASSF1A promoter region CpG island methylation was detected in 72% of SCLC, 34% of NSCLC, 9% of breast, 10% of ovarian and 0% of primary cervical tumours and in 72% SCLC, 36% NSCLC, 80% of breast and 40% of ovarian tumour lines. In view of the lower frequency of RASSF1 methylation in primary breast cancers we proceeded to RASSF1 mutation analysis in 40 breast cancers. No mutations were detected, but six single nucleotide polymorphisms were identified. Twenty of 26 SCLC tumours with 3p21.3 allelic loss had RASSF1A methylation, while only six out of 22 NSCLC with 3p21.3 allele loss had RASSF1A methylation (P=0.0012), one out of five ovarian and none out of six cervical tumours with 3p21.3 loss had RASSF1A methylation. These results suggest that (a) RASSF1A inactivation by two hits (methylation and loss) is a critical step in SCLC tumourigenesis and (b) RASSF1A inactivation is of lesser importance in NSCLC, breast, ovarian and cervical cancers in which other genes within LCTSGR1 are likely to be implicated.

Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12(DOC-1) is a growth suppressor isolated from normal keratinocytes. We report that p12(DOC-1) associates with CDK2. More specifically, p12(DOC-1) associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12(DOC-1) resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12(DOC-1) transfectants ( upward arrow G(1) and downward arrow S). The p12(DOC-1)-mediated decrease of CDK2 was prevented if the p12(DOC-1) transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin beta-lactone, suggesting that p12(DOC-1) may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12(DOC-1)-mediated, CDK2-associated cell cycle phenotypes. These data support p12(DOC-1) as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.

We have positionally cloned and characterized a new calcium channel auxiliary subunit, alpha(2)delta-2 (CACNA2D2), which shares 56% amino acid identity with the known alpha(2)delta-1 subunit. The gene maps to the critical human tumor suppressor gene region in chromosome 3p21.3, showing very frequent allele loss and occasional homozygous deletions in lung, breast, and other cancers. The tissue distribution of alpha(2)delta-2 expression is different from alpha(2)delta-1, and alpha(2)delta-2 mRNA is most abundantly expressed in lung and testis and well expressed in brain, heart, and pancreas. In contrast, alpha(2)delta-1 is expressed predominantly in brain, heart, and skeletal muscle. When co-expressed (via cRNA injections) with alpha(1B) and beta(3) subunits in Xenopus oocytes, alpha(2)delta-2 increased peak size of the N-type Ca(2+) currents 9-fold, and when co-expressed with alpha(1C) or alpha(1G) subunits in Xenopus oocytes increased peak size of L-type channels 2-fold and T-type channels 1.8-fold, respectively. Anti-peptide antibodies detect the expression of a 129-kDa alpha(2)delta-2 polypeptide in some but not all lung tumor cells. We conclude that the alpha(2)delta-2 gene encodes a functional auxiliary subunit of voltage-gated Ca(2+) channels. Because of its chromosomal location and expression patterns, CACNA2D2 needs to be explored as a potential tumor suppressor gene linking Ca(2+) signaling and lung, breast, and other cancer pathogenesis. The homologous location on mouse chromosome 9 is also the site of the mouse neurologic mutant ducky (du), and thus, CACNA2D2 is also a candidate gene for this inherited idiopathic generalized epilepsy syndrome.

Kidney cancer affects nearly 28,000 Americans each year and is responsible for 12,000 deaths annually in the United States. Kidney cancer can occur as localized, locally advanced, or advanced disease. Little is known about the specific cause of kidney cancer, although as with smoking, a number of associations have been noted. Kidney cancer can occur in both a hereditary as well as a nonhereditary sporadic form [1]. When two or more members of the same family are found to have kidney cancer, screening of other at-risk individuals in the family is recommended.

A number of genes underlie the molecular bases of intelligence. Among these is probably CALL, a novel member of the L1 gene family of neural cell adhesion molecules. By using the single strand conformation polymorphism (SSCP) protocol, we screened the regions of the CALL gene corresponding to the 5' and 3' untranslated regions (UTRs) of the CALL mRNA, searching for polymorphisms that could be useful in association studies in the field of intelligence. We report the finding of T-to-A and T-to-C single nucleotide polymorphisms (SNPs) in the 3' UTR of CALL. These SNPs have an index of heterozygosity of 0.13 and 0.10, respectively. Research is in progress to understand the association between these variants and high IQ.