Suppression of murine mammary carcinoma metastasis by the murine ortholog of breast cancer metastasis suppressor 1 (Brms1)

The murine ortholog (Brms1) of human breast cancer metastasis suppressor 1 shares 95% identity to the human metastasis suppressor, BRMS1, in amino acid structure. We tested Brms1 for suppression of metastasis of mouse mammary carcinoma cell line 4T1 in syngenic BALB/c mice, using orthotopic (mammary fat pad) injection as well as intravenous injection. As observed for BRMS1, transfection with Brms1 did not inhibit 4T1 primary tumor formation, but significantly suppressed lung colonization. We also show that Brms1 protein interacts with histone deacetylases, indicating involvement of Brms1 in murine Sin3-HDAC complex, like its human counterpart. Thus, because of similarities with its human ortholog, the results suggest that Brms1 will be useful as a model for studying mechanism of action of BRMS1.     

Disruption of the RanBP17/Hox11L2 region by recombination with the TCRdelta locus in acute lymphoblastic leukemias with t(5;14)(q34;q11).

The t(5;14)(q33-34;q11) translocation constitutes a recurrent rearrangement in acute lymphoblastic leukemia involving the T cell receptor (TCR) delta locus on chromosome 14. Breakpoint sequences of the derivative chromosome 5 were isolated by application of a ligation-mediated PCR technique using TCR delta-specific primers to amplify genomic DNA from the leukemic cells of a patient with t(5;14). Through exon trap analysis, we identified various putative exons of the chromosome 5 target gene of the translocation; compilation of sequence information of trapped exons and available expressed sequence tags (ESTs) from the GenBank database allowed us to assemble 1.2 kb of the cDNA. Full-length cDNAs were isolated from a human testis cDNA library and sequence analysis predicted a putative Ran binding protein, a novel member of the importin-beta superfamily of nuclear transport receptors, called RanBP17. The t(5;14) breakpoint maps to the 3' coding region of the gene. The breakpoint of a second t(5;14) positive patient was mapped about 8 kb downstream of the most 3' RanBP17 exon and 2 kb upstream of the first exon of the orphan homeobox gene, Hox11L2. In both cases TCR delta enhancer sequences are juxtaposed downstream of the truncated or intact RanBP17 gene, respectively on the derivative chromosome.     

Isolation and characterization of cDNA and genomic sequences for mouse O6-methylguanine-DNA methyltransferase.

An enzyme, O6-methylguanine-DNA methyltransferase, is present in various organisms and plays an important role in repair of DNA damaged by alkylating agents. The enzyme transfers methyl groups from O6-methylguanine and other methylated moieties of the DNA to its own molecule. As a first step to construct animal models with altered levels of the enzyme activity, we cloned cDNA and genomic DNA sequences for mouse methyltransferase and elucidated their structures. The nucleotide sequence of the cDNA revealed an open reading frame comprising 211 amino acid residues. The mol. wt of mouse O6-methylguanine-DNA methyltransferase, calculated from the predicted amino acid sequence, was 22,400, and the methyltransferase protein of this size was present when the cDNA was expressed in methyltransferase-deficient human cells. The predicted amino acid sequence of the mouse methyltransferase exhibits an intense homology with those of human and bacterial counterparts. Using the cDNA as a probe, part of the mouse gene for methyltransferase was isolated. The gene consisted of at least four exons and spanned greater than 145 kb. Sequences around the exon/intron junctions for the mouse gene are almost the same as those for the human species.     

High level of messenger RNA for BRMS1 in primary breast carcinomas is associated with poor prognosis

BRMS1 is regarded as a metastasis suppressor gene for its ability to reduce metastatic potential of human and murine breast cancer cells as well as human melanoma cells. However, BRMS1 association to human tumor progression is not clearly understood. In the present study we analyzed BRMS1 mRNA expression in tumor progression and its potential prognostic value for breast carcinoma. BRMS1 mRNA expression level was quantified by real-time PCR in 47 tumoral, in 14 peritumoral and in 15 metastatic microdissected cellular populations from 47 breast cancer patients with 10-year follow up. We found BRMS1 expression to be higher in carcinoma cells than in matching normal epithelial cell populations in 10 out of 14 cases (p = 0.0005), while lymph-nodal carcinoma cells showed lower BRMS1 expression in 9 out of 15 cases (p = 0.001). Using both in vivo (human mammary breast carcinomas) and in vitro systems (breast cancer cell lines) we were able to demonstrate that BRMS1 overexpression was not a bias effect induced by cell proliferation rate. BRMS1 expression levels did not correlate with standard breast cancer prognostic factors but BRMS1 higher expression was associated with patient shorter disease-free and overall survival. Our findings are apparently inconsistent with the concept of BRMS1 as a metastasis suppressor gene. One possible explanation is that epithelial cells increase their BRMS1 expression as a compensatory response to tumor formation or metastasis progression, which is elevated in proportion to tumor aggressiveness, whereas those cells of the primary tumor that cannot upregulate BRMS1 escape to form metastasis.     

人UREB1基因克隆及其在肿瘤组织中的分布

目的：大量UREB基因编码的蛋白质能特异地结合位于强吗啡基因启动子上游的一段DNA序列（upstream regulatory element,URE)。早期研究证实UREB1对强吗啡基因启动子的转录具有促进作用,而对p53的转录激活作用具有抑制效应。本实验目的的就是克隆人UREB1基因,探索其与肿瘤发生发展的关系。方法：利用人工合成寡核苷酸探针从人脑cDNA文库中筛选人UREB1基因,应用大肠杆菌表达的重组蛋白质制备的抗体检测肿瘤组织中UREB1的表达分布。结果：获得了人UREB1基因,核苷酸序列在对应区域及氨基酸序列与大鼠UREB1基因cDNA序列和氨基酸序列皆有91％的同源性。在各种肿瘤组织中都有UREB1的表达,但是表达水平及定位不一样。初步发现这样一个规律,随着肿瘤的恶性程度增加UREB1在核内的聚集程度增加。UREB1的酪氨酸磷酸化分析结果显示,肿瘤恶性程度高,UREB1的酪氨酸磷酸化程度高。结论：UREB1可能参与肿瘤的发生发展,其酪氨酸磷酸化水平可以影响肿瘤的恶性程度。     

Structures and comparison of genomic and complementary DNAs of mouse MSSP, a c-Myc binding protein

Two cDNAs encoding mouse MSSP and Scr3 were cloned. Both proteins, like those in humans, share significant homology of the region near the N-terminus containing the RNA-binding protein, RNP, while little homology exists near the C-terminal region of the proteins. Expression of mouse MSSP and Scr3 in mouse tissues were examined by Northern blot hybridization and both mRNAs were ubiquitously expressed in all the tissues except for testis, where the smaller sizes of MSSP mRNAs, but not Scr3 mRNA, were highly expressed. Then, the genomic DNA of mouse MSSP was cloned. Mouse genomic MSSP gene was comprised of at least 13 exons over the region spanning 60 kb. Furthermore, a chromosome location of human MSSP gene was identified at 2q24 by FISH mapping.     

Molecular cloning of human Hic-5, a potential regulator involved in signal transduction and cellular senescence

By using differential display, we cloned the human counterpart of the murine gene Hic-5 from senescent human keratinocytes. The full-length cDNA contained a short GC-stretch proceeding a consensus Kozak sequence followed by a single open reading frame of 1338 bp encoding a 461-amino acid protein with a predicted molecular weight of 50 kDa. The expression of this gene was prominent in cells of epithelial origin but low or absent in lymphoid tissues and hematopoietic cells. The deduced protein contained four LIM domains at the carboxyl-terminal end and four LD motifs at the amino-terminal half, sharing high similarities with the focal adhesion protein paxillin. Hic-5 may therefore function, like paxillin, as a potential adapter for the recruitment of structural and signaling molecules to certain subcellular sites or in focal adhesions. Isolation of the genomic sequence revealed that the gene covered a segment of 6 kb and spanned 11 exons from the translation initiation site ATG to the termination signal TGA. Fluorescent in situ hybridization by using a human Hic-5 specific probe localized the gene to human chromosome 16p11.     

The Murine cot Proto-oncogene: Genome Structure and Tissue-specific Expression

We cloned and analyzed the murine cot proto-oncogene and examined its tissue-specific expression in fetal, newborn and adult mice. Genomic cot DNA consists of eight exons, spanning more than 25 kb, and all intron-exon borders are well conserved as compared to the human homolog. Analysis of the full-length cot cDNA revealed that it contained an open reading frame of 1,401 nucleotides, like human cot proto-oncogene. The sequence identity between murine and human cot gene is 84.4% at the nucleotide level and 93.9% at the deduced amino acid level. On northern blot analysis of poly (A)⁺ RNA, the cot message was detected at 2.9 kb in size. Expression of the cot gene was observed in many tissues from fetal to adult mice, though the level of expression was low in all tissues examined.     

cDNA and genomic sequences for rat 8-oxo-dGTPase that prevents occurrence of spontaneous mutations due to oxidation of guanine nucleotides

The enzyme, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase),is present In various organisms and plays an important rolein control of spontaneous mutagenesis. This enzyme degrades8-oxoguamne-contaning deoxyribonucleoside triphosphate, a potentiallymutagenic substrate for DNA synthesis, to the correspondingmonophosphate. To obtain appropriate probes for expression ofthe gene in various tissues and also to construct appropriateexperimental models for carcino genesis, we cloned cDNA forrat 8-oxo-dGTPase and elucidated its structure. The nucleotidesequence of the cDNA revealed that the rat 8-oxo-dGTPase proteinis composed of 156 amino acid residues. The molecular weightof rat 8-oxo-dGTPase, calculated from the predicted amino acidse was 18 006, and the 8-oxo-dGTPase protein of this size wasdetected when the cDNA was expressed in 8-oxo-dGTPase-deficlentEscherichitz coil mutT^– cells. The predicted amino acidsequence of the rat 8-oxo-dGTPase has a close homology withthose of human and bacterial counterparts. Using the cDNA asa probe, part of the rat gene for 8-oxo-dGTPase was isolatedand was found to consist of at least three exons and spannedabout 10 kb. A genomic region containing the pseudogene wasalso isolated.     

The human homologue of the putative proto-oncogene Spi-1: characterization and expression in tumors.

Spi-1 is a putative proto-oncogene involved in murine virus-induced acute erythroleukemias. We report here the identification of the human homologue of Spi-1 and its expression in normal and tumorigenic human tissues. Characterization of cDNA clones revealed that the human Spi-1 gene encodes a 216 amino acids protein showing 85% identity with the murine counterpart. By sequencing genomic clones, five exons were identified. To investigate the possible role of Spi-1 gene in human cancers, we studied its expression in a panel of human tumors by Northern blot analysis. Spi-1 expression was detected in all the tumors examined. There was no noticeable evidence of messenger RNA alteration as compared to normal tissues.     

Human hst-2 (FGF-6) oncogene: cDNA cloning and characterization.

The hst-2 gene was previously identified by its close homology to the hst-1 gene. Cosmid clones containing the hst-2 gene were cloned from a normal human genomic library. Focus-forming activity was observed for the hst-2 cosmids when NIH3T3 transfection assay was performed in a serum-free medium, whereas induction of morphological transformation was difficult to detect in an ordinary serum-supplemented medium. The hst-2 cDNA was cloned from the NIH3T3 transformant. Nucleotide sequence analysis of the cDNA indicates that the hst-2 gene encodes a 198 amino acid transforming protein containing a signal peptide with the characteristics of a heparin-binding growth factor. The coding sequence was almost identical to the published portion of the exon sequence of the FGF-6 gene, indicating that hst-2 is identical to FGF-6. The hst-2 cDNA fragment, when inserted into an expression vector, was able to transform NIH3T3 cells effectively, and the resulting transformant formed a well-vascularized tumor in nude mice, thus suggesting an angiogenic property similar to some other members of the family. RNA blot analysis revealed the expression of the hst-2 gene in human leukemia cell lines with platelet/megakaryocytic differentiation potential.