Severe suppression of Frzb/sFRP3 transcription in osteogenic sarcoma

Deciphering the molecular basis of cancer is critical for developing novel diagnostic and therapeutic strategies. To better understand the early molecular events involving osteogenic sarcoma (OGS), we have initiated a program to identify potential tumor suppressor genes. Expression profiling of total RNA from ten normal bone cell lines and eleven OGS-derived cell lines by microarray showed 135-fold lower expression of FRZB/sFRP3 mRNA in OGS cells compared to bone cells; this down-regulation of Frzb/sFRP3 mRNA expression was found to be serum-independent. Subsequently, fourteen OGS biopsy specimens showed nine-fold down-regulation of Frzb/sFRP3 mRNA expression compared to expression in eight normal bone specimens as determined by microarray. FRZB /sFRP3 protein level was also found to be at a very low level in 4/4 OGS cell lines examined. Quantitation by RT-PCR indicated approximately 70% and approximately 90% loss of Frzb/sFRP3 mRNA expression in OGS biopsy specimens and OGS-derived cell lines respectively, compared to expression in bone (p<0.0001). Hybridization experiments of a cDNA microarray containing paired normal and tumor specimens from nineteen different organs did not show any significant difference in the level of Frzb/sFRP3 mRNA expression between the normal and the corresponding tumor tissues. Exogenous expression of FRZB/sFRP3 mRNA in two OGS-derived cell lines lacking endogenous expression of the mRNA produced abundant mRNA from the exogenous gene, eliminating degradation as a possibility for very low level of FRZB/sFRP3 mRNA in OGS specimens. Results from PCR-based experiments suggest that the FRZB/sFRP3 gene is not deleted in OGS cell lines, however, karyotyping shows gross abnormalities involving chromosome 2 (location of the FRZB gene) in five of twelve OGS-derived cell lines. Together, these data suggest a tumor-suppressive potential for FRZB/sFRP3 in OGS.     

High expression of tumor endothelial marker 7 is associated with metastasis and poor survival of patients with osteogenic sarcoma

Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metastasis is the primary cause of mortality among patients with OGS. To identify such genes, we first created a database of differentially expressed genes between six low-grade and six high-grade OGS tumors, and between a normal immortalized osteoblast cell line (FOB) and four commercially available OGS-derived cell lines. We specifically searched for surface proteins over-expressed in high-grade OGS, since we hypothesize that tumor-cell specific surface markers are key to metastasis. A gene encoding Tumor Endothelial Marker7 (TEM7) was selected as a candidate for further study. TEM7 expression pattern was assessed by RT-PCR, Western blotting and immunostaining. TEM7 mRNA was abundantly expressed in SAOS cells (derived from high-grade OGS), but not in FOB or MG63 cells (derived from low-grade OGS). Virtually no expression of TEM7 protein was observed in FOB cells but abundant expression was noted in SAOS and TE85 cells. Employing immunostaining of 92 human OGS specimens (50 high-grade and 42 low-grade) collected before chemotherapy show 97% (37 of 38) of high-grade OGS specimens with metastasis have high TEM7 staining. Further, we found that elevated expression of TEM7 correlated with poor survival (p<0.04) of affected patients. Inhibiting TEM7 function by siRNA inhibited invasion and migration of OGS cells with metastatic potential. Our results suggest TEM7 expression level closely parallels histology-based prognostication of OGS metastasis and, therefore, it is a therapeutic target. This is the first demonstration of a link between TEM7 and cancer metastasis.     

Cloning and Characterization of HumanSep1 (hSEP1) Gene and Cytoplasmic Localization of its Product

We isolatedand sequenced a human cDNA (designated as hSEP1)encoding both a homologue of mouse Dhm2 and budding yeast SEP1.The gene was shown to be locatedon the long arm of chromosome3 (3q25-26.1). The putative hSEP1 product (hSEP1p) consistedof 1694 amino acid residues with a molecular mass of about 190kDa. Northern blot analysis showeda major 10-kb mRNA expressedubiquitously in variousorgans as well as a minor 5.5-kb mRNAexpressed relatively highly in the testis and placenta. hSEP1pis localizedin the cytoplasm as examinedb y cytochemical andWestern blot analyses of fractionated cellular extracts. Thebiological function of hSEP1p was discussed in correlation withits cytoplasmic localization.     

Cloning a calcium channel alpha2delta-3 subunit gene from a putative tumor suppressor gene region at chromosome 3p21.1 in conventional renal cell carcinoma

We have identified loss of heterozygosity (LOH) of approx. 1 cM region around locus D3S1289 at chromosome 3p21.1 in a conventional renal cell carcinoma (RCC). During construction of a YAC/BAC contig for this region and shotgun sequencing of BACs 277p5, 55m24 and 428i24, we detected four new microsatellites. We narrowed down the target region by analysing these new loci to less than 100 kb within the BAC 55m24 and subsequently cloned a human calcium channel alpha2delta-3 subunit gene. This gene is widely expressed in fetal tissues and different types of adult tumors. The exons of the alpha2delta-3 subunit gene are distributed along approx. 500 kb DNA sequences. As the LOH involved exclusively intronic sequences and sequencing the entire coding region did not reveal any mutation, the alpha2delta-3 subunit gene is probably not a tumor suppressor gene.     

Effect of 1,25(OH)2D3 on bone morphogenetic protein-3 mRNA expression

Bone morphogenetic proteins (BMPs) are members to the transforming growth factor-beta superfamily. They induce ectopic bone formation in rat and are pleiotropic initiators of inducible osteogenic precursor cells. A lot of reports have studied the presence of BMPs and their effects on bone marker expression in many different cell lines, however none describe the regulation of BMP3 by different factors and expression conditions. When a human bone marrow stromal cell (HBMSC) culture was treated simultaneously with 1,25(OH)2D3 (10(-8) M) and BMP3 (2.5 ng/ml), the total osteocalcin content in the cell layer and in the culture medium was higher than when the culture was treated with either factor alone (162%). To elucidate this synergistic activity, Northern blot analysis was done to study the effect of 1,25(OH)2D3 on BMP3 mRNA expression. Several human cell lines (MNNG, U-2OS, MG-63, KHOS, TE85, HOS) and HBMSC were treated by 1,25(OH)2D3 (10(-8) M for 24 h). Purified mRNA from treated and untreated cells were denatured using glyoxal and dimethylsulfoxide, and were fractionated on a 1% agarose gel. After electrophoresis, RNA were blotted onto a nylon membrane and incubated with 32P-labeled BMP3 and GAPDH riboprobes. Northern blot analysis revealed that, the BMP3 mRNA level was increased in a few cell lines (MG-63, HBMSC, HOS) after the addition of 1,25(OH)2D3 when compared to the untreated cells (127%+/-1; 130.5%+/-19.5; 207%+/-14). An higher stimulation was observed in HBMSC primary culture when compared to differentiated HBMSC. In view of these results, we now investigate the following hypothesis: does the BMP3 promoter exhibit the vitamin D receptor response like the osteocalcin gene?     

Comparative genomic hybridization study of nasal-type NK/T-cell lymphoma

BACKGROUND: Nasal-type NK/T-cell lymphoma is a rare type of non-Hodgkin's lymphoma. The genetic changes associated with pathogenesis have not been well defined. This study investigates the nonrandom genetic alteration of nasal-type NK/T-cell lymphoma. METHODS: Nine cases were studied. Comparative genomic hybridization (CGH) was carried out using fresh tumor tissues of seven nasal-type NK/T-cell lymphomas. To complement the data by CGH, loss of heterozygosity (LOH) of chromosomes 6q, 1p, and 17p using polymorphic markers and p53 gene mutation by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) were analyzed. RESULTS: The DNA copy number changes of seven nasal-type NK/T-cell lymphomas were gains on chromosomes 2q(5), 13q(4), 10q(3), 21q(2), 3q(2), 5q(2), and 17q(2), and losses involving chromosomes 1p(4), 17p(4), 12q(3), 13q(2), and 6q(1). One of six cases informative for at least two markers for chromosome 6q showed LOH at D6S300, D6S1639, D6S261, D6S407, and D6S292. Two cases showing loss of 1p and 17q by CGH revealed LOH at D1S214, D1S503, and D17S559. P53 mutation was detected in exon 8 in one of nine cases. CONCLUSIONS: Frequent DNA losses at 1p, 17p, and 12q and gains at 2q, 13q, and 10q suggested that these regions could be targets for further molecular genetic analysis to investigate tumor suppressor genes or oncogenes associated with tumorigenesis of NK/T-cell lymphoma. Infrequent alteration of 6q contrary to previous studies raises doubt about an implication of 6q loss in the pathogenesis of early-stage NK/T-cell lymphoma. Further studies on more defined cases are required to verify their association.     

An 800-kb region of deletion at 13q14 in human prostate and other carcinomas

Deletions of regions at 13q14 have been detected by various genetic approaches in human cancers including prostate cancer. Several studies have defined one region of loss of heterozygosity (LOH) at 13q14 that seems to reside in a DNA segment of 7.1 cM between genetic markers D13S263 and D13S153. To define the smallest region of overlap (SRO) for deletion at 13q14, we first applied tissue microdissection and multiplex PCR to detect homozygous deletion and/or hemizygous deletion at 13q14 in 134 prostate cancer specimens from 114 patients. We detected deletions at markers D13S1227, D13S1272, and A005O48 in 13 (10%) of these tumor specimens. Of the 13 tumors with deletions, 12 were either poorly differentiated primary tumors or metastases of prostate cancer. To fine-map the deletion region, we then constructed a high-resolution YAC/BAC/STS/EST physical map based on experimental and database analyses. Several markers encompassing the deletion region were analyzed for homozygous deletion and/or hemizygous deletion in 61 cell lines/xenografts derived from human cancers of the prostate, breast, ovary, endometrium, cervix, and bladder, and a region of deletion was defined by duplex PCR assay between markers A005X38 and WI-7773. We also analyzed LOH at 13q14 in the 61 cell lines/xenografts using the homozygosity mapping of deletion approach and 26 microsatellite markers. We found 24 (39%) of the cell lines/xenografts to show LOH at 13q14 and defined a region of LOH by markers M1 and M5. Combination of homozygous or hemizygous deletion and LOH results defined the SRO for deletion to be an 800-kb DNA interval between A005X38 and M5. There are six known genes located in or close to the SRO for deletion. This region of deletion is at least 2 Mb centromeric to the RB1 tumor-suppressor gene and the leukemia-associated genes 1 and 2, each of which is located at 13q14. These data suggest that the 800-kb DNA segment with deletion contains a gene whose deletion may be important for the development of prostate and other cancers. This study also provides a framework for the fine-mapping, cloning, and identification of a novel tumor-suppressor gene at 13q14.     

A novel,evolutionarily conserved gene family with putative sequence-specific single-stranded DNA-binding activity

Complete and partial deletions of chromosome 5q are recurrent cytogenetic anomalies associated with aggressive myeloid malignancies. Earlier, we identified an approximately 1.5-Mb region of loss at 5q13.3 between the loci D5S672 and D5S620 in primary leukemic blasts. A leukemic cell line, ML3, is diploid for all of chromosome 5, except for an inversion-coupled translocation within the D5S672-D5S620 interval. Here, we report the development of a bacterial artificial chromosome (BAC) contig to define the breakpoint and the identification of a novel gene SSBP2, the target of disruption in ML3 cells. A preliminary evaluation of SSBP2 as a tumor suppressor gene in primary leukemic blasts and cell lines suggests that the remaining allele does not undergo intragenic mutations. SSBP2 is one of three members of a closely related, evolutionarily conserved, and ubiquitously expressed gene family. SSBP3 is the human ortholog of a chicken gene, CSDP, that encodes a sequence-specific single-stranded DNA-binding protein. SSBP3 localizes to chromosome 1p31.3, and the third member, SSBP4, maps to chromosome 19p13.1. Chromosomal localization and the putative single-stranded DNA-binding activity suggest that all three members of this family are capable of potential tumor suppressor activity by gene dosage or other epigenetic mechanisms.     

Mechanisms of loss of heterozygosity in retinoblastoma.

Retinoblastoma (RB) tumors arise when both alleles of the RB1 gene are inactivated by two mutational events (M1 and M2). M1 can be an initial germline or somatic mutation; M2 is frequently loss of heterozygosity (LOH), which makes the cell homozygous or hemizygous for the original mutation. LOH is the major mechanism by which many cancers are initiated. To further delineate the mechanism of LOH, we screened a total of 37 RB tumors for LOH by Southern blot analysis. The tumors were from 17 bilaterally and 17 unilaterally affected patients. Nineteen of 30 informative tumors (63%) from 27 patients showed LOH. Proximal and distal flanking markers on chromosome 13 were informative in 13 tumors, allowing evaluation of the mechanisms by which LOH occurred. Mitotic recombination was implicated in 6 (46%) of the 13 tumors.     

In vitro and in vivo studies of antiosteosarcoma activities of formononetin

Osteogenic sarcoma (OGS) is a primary bone cancer, characterized by aggressive neoplasm from mesenchymal oncogenesis. However, the clinical therapeutic regimen against OGS is limited. Therefore, potential medication warrants to be further developed. Our previous study indicates that formononetin (FN) exerts effective pharmacological activity against OGS. This study aimed to further decipher the molecular mechanism behind this benefit. Patients with OGS were recruited for clinical data assay and immunoassay. Human OGS cell line (U2OS) and tumor-bearing nude mice were subjected to a battery of biochemical analyses and immunoassays for integrative evaluation of FN-exerted anti-OGS effects. In human data, OGS samples showed increased expressions of ERα, p-PI3KCA^Tyr317, and p-AKT ^Ser473 proteins, followed by notably upregulated miR-375 content in comparison with that in OGS-free. In addition, FN-treated U2OS cells showed inhibited cell proliferation, elevated lactic dehydrogenase production and lowered endogenous miR-375 level in cells. Further, reduced immunopositive cells of Ki-67, p-PI3KCA ^Tyr317, and p-AKT ^Ser473 were observed by the treatments of FN, while the intracellular Bax- and Apaf-1-positive cells were increased dose-dependently. Beneficially, FN-treated tumor-bearing mice exhibited reduced tumor mass and intercellular miR-375 expression. Meanwhile, immuno-labeled cells and proteins of Bax, Caspase-3, and Apaf-1 in FN-treated mice were increased dose-dependently, whereas ERα, p-PI3KCA ^Tyr317, and p-AKT ^Ser473 positive cells and proteins were downregulated, respectively. Collectively, our current results elucidate that FN exerts effective therapeutic benefits against OGS, and the pharmacological mechanism may be related to promoting cell apoptosis by inactivating intracellular miR-375/ERα−PI3K/AKT cascaded pathway.     

Loss of NOTCH2 positively predicts survival in subgroups of human glial brain tumors

The structural complexity of chromosome 1p centromeric region has been an obstacle for fine mapping of tumor suppressor genes in this area. Loss of heterozygosity (LOH) on chromosome 1p is associated with the longer survival of oligodendroglioma (OD) patients. To test the clinical relevance of 1p loss in glioblastomas (GBM) patients and identifiy the underlying tumor suppressor locus, we constructed a somatic deletion map on chromosome 1p in 26 OG and 118 GBM. Deletion hotspots at 4 microsatellite markers located at 1p36.3, 1p36.1, 1p22 and 1p11 defined 10 distinct haplotypes that were related to patient survival. We found that loss of 1p centromeric marker D1S2696 within NOTCH2 intron 12 was associated with favorable prognosis in OD (P = 0.0007) as well as in GBM (P = 0.0175), while 19q loss, concomitant with 1p LOH in OD, had no influence on GBM survival (P = 0.918). Assessment of the intra-chromosomal ratio between NOTCH2 and its 1q21 pericentric duplication N2N (N2/N2N-test) allowed delineation of a consistent centromeric breakpoint in OD that also contained a minimally lost area in GBM. OD and GBM showed distinct deletion patterns that converged to the NOTCH2 gene in both glioma subtypes. Moreover, the N2/N2N-test disclosed homozygous deletions of NOTCH2 in primary OD. The N2/N2N test distinguished OD from GBM with a specificity of 100% and a sensitivity of 97%. Combined assessment of NOTCH2 genetic markers D1S2696 and N2/N2N predicted 24-month survival with an accuracy (0.925) that is equivalent to histological classification combined with the D1S2696 status (0.954) and higher than current genetic evaluation by 1p/19q LOH (0.762). Our data propose NOTCH2 as a powerful new molecular test to detect prognostically favorable gliomas.     

Saccharomyces cerevisiae proteins involved in hybrid DNA formation in vitro

RecA-like activities that can form hybrid DNA in vitro have been identified in a wide variety of organisms. We have previously described the strand exchange protein 1 (SEP1) from the yeast Saccharomyces cerevisiae that can form hybrid DNA in vitro. Purified as an Mr 132,000 polypeptide, recent molecular and immunological studies have now shown that the native form is an Mr 175,000 polypeptide containing strand exchange activity. The gene encoding SEP1 has been cloned and sequenced. The primary sequence failed to reveal any significant sequence homology to other sequences in data base searches. In vivo SEP1 was found to be essential for normal meiosis as cells containing a homozygous insertion mutation in the SEP1 gene failed to sporulate. In order to identify additional factors that are involved in hybrid DNA formation in S cerevisiae, we used an in vitro stimulation assay to identify proteins that reconstitute strand exchange activity in reactions containing limiting amounts of SEP1. We have identified two proteins that functionally interact with SEP1. First, an Mr 34,000 single-stranded DNA binding protein stimulated the reaction by lowering the requirement for SEP1 about 3-4 fold. This protein is a fragment of the large subunit of a hetero-trimeric complex called yRP-A (yRF-A) which is thought to be the functional eukaryotic equivalent of single-stranded DNA binding proteins in prokaryotes. The gene encoding this protein (RPA1) is essential for growth. Second, an Mr 33,000 polypeptide, termed Stimulatory Factor 1 (SF1), dramatically stimulated the SEP1 catalyzed reaction by lowering the requirement for SEP1 about 300 fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine mapping of the NRC-1 tumor suppressor locus within chromosome 3p12

Identification of tumor suppressor genes based on physical mapping exercises has proven to be a challenging endeavor, due to the difficulty of narrowing regions of loss of heterozygosity (LOH), infrequency of homozygous deletions, and the labor-intensive characterization process for screening candidates in a given genomic interval. We previously defined a chromosome 3p12 tumor suppressor locus NRC-1 (Nonpapillary Renal Carcinoma-1) by functional complementation experiments in which renal cell carcinoma microcell hybrids containing introduced normal chromosome 3p fragments were either suppressed or unsuppressed for tumorigenicity following injection into athymic nude mice. We now present the fine-scale physical mapping of NRC-1 using a QPCR-based approach for measuring copy number at sequence tagged sites (STS) which allowed a sub-exon mapping resolution. Using STS-QPCR and a novel statistical algorithm, the NRC-1 locus was narrowed to 4.615-Mb with the distal boundary mapping within a 38-Kb interval between exon 3 and exon 4 of the DUTT1/Robo1 gene, currently the only candidate tumor suppressor gene in the interval. Further mutational screening and gene expression analyses indicate that DUTT1/ROBO1 is not involved in the tumor suppressor activity of NRC-1, suggesting that there are at least two important tumor suppressor genes within the chromosome 3p12 interval.     

Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.     

An apparent interlocus gene conversion-like event at a putative tumor suppressor gene locus on human chromosome 6q27 in a Burkitt's lymphoma cell line.

A region of minimal deletion in B-cell non-Hodgkin's lymphoma (B-NHL) has recently been defined between D6S186 and D6S227 spanning 5-9 Mb at 6q26-q27, predicting the presence of at least one tumor suppressor gene (TSG) at this locus. During the construction of a deletion map in the B-NHL tumor panel, we report the identification of a Burkitt's lymphoma cell line, BL74, having an apparent homozygous deletion at the D6S347 locus, internal to the critical region. Since this case may facilitate the localization of the target TSG, a detailed structural molecular characterization and search for candidate genes were undertaken at this locus. While BL74 underwent a loss of heterozygosity at 6q26-q27, D6S347 was also likely subjected to a somatic interlocus gene conversion-like event between two homologous but distinct loci, resulting in the homozygous replacement of a 1860- to 2067-bp segment of one locus with the corresponding segment copied from the other locus. Two genes at this locus were identified, but their lack of expression in B-cell lineages tentatively excludes them as candidate TSGs. Another still unidentified gene at this locus may be disrupted by the gene conversion-like event, which would represent a novel mechanism of TSG inactivation.     

Physical Mapping of a Commonly Deleted Region, the Site of a Candidate Tumor Suppressor Gene, at 12q22 in Human Male Germ Cell Tumors

A candidate tumor suppressor gene (TSG) site at 12q22 characterized by a high frequency of loss of heterozygosity (LOH) and a homozygous deletion has previously been reported in human male germ cell tumors (GCTs). In a detailed deletion mapping analysis of 67 normal-tumor DNAs utilizing 20 polymorphic markers mapped to 12q22–q24, we identified the limits of the minimal region of deletion at 12q22 between D12S377 (proximal) and D12S296 (distal). We have constructed a YAC contig map of a 3-cM region of this band between the proximal marker D12S101 and the distal marker D12S346, which contained the minimal region of deletion in GCTs. The map is composed of 53 overlapping YACs and 3 cosmids onto which 25 polymorphic and nonpolymorphic sequence-tagged sites (STSs) were placed in a unique order. The size of the minimal region of deletion was approximately 2 Mb from overlapping, nonchimeric YACs that spanned the region. We also developed a radiation hybrid (RH) map of the region between D12S101 and D12S346 containing 17 loci. The consensus order developed by RH mapping is in good agreement with the YAC STS-content map order. The RH map estimated the distance between D12S101 and D12S346 to be 246 cR₈₀₀₀and the minimal region of deletion to be 141 cR₈₀₀₀. In addition, four genes that were previously mapped to 12q22 have been excluded as candidate genes. The leads gained from the deletion mapping and physical maps should expedite the isolation and characterization of the TSG at 12q22.