Cloning and characterization of a novel gene,WS-3, in human chromosome 8p11-p12

A novel human gene referred to as the WS-3 gene, in the short arm of human chromosome 8, was cloned by a combination of exon trapping, thermal asymmetric interlaced-PCR (TAIL-PCR) and the Marathon-Ready cDNA amplification method. The gene consists of 7 exons separated by 6 introns, and is at the telomere side of the STS marker, D8S1055. The full-length WS-3 gene contains 1052 nucleotides and codes for a protein of 190 amino acids with a calculated mol. wt. of 20 747. Southern blot experiments showed that the WS-3 gene exists as a single copy in the human genome. A protein encoded by the WS-3 gene has an R-G-D (Arg-Gly-Asp) motif in the N-terminal region, which seems to confer adhesive properties to macromolecular proteins like fibronectin. Although WS-3 is a small gene with unknown biological function, its ubiquitous expression in various tissues and organs suggests that the encoded protein is one of the essential components of all organs and tissues.     

KLK12 is a novel serine protease and a new member of the human kallikrein gene family-differential expression in breast cancer

Kallikreins are a subgroup of serine proteases that are involved in the posttranslational processing of polypeptide precursors. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins are encoded by a large multigene family, but in humans, only three genes have been identified. By using the positional candidate approach, we were able to identify a new kallikrein-like gene, tentatively named KLK12 (for kallikrein gene 12). This new gene maps to chromosome 19q13.3-q13.4, is formed of five coding exons, and shows structural similarity to serine proteases and other known kallikreins. KLK12 is expressed in a variety of tissues including salivary gland, stomach, uterus, lung, thymus, prostate, colon, brain, breast, thyroid, and trachea. We identified three splicing forms of KLK12 that are expressed in many tissues. Our preliminary results indicate that the expression of KLK12 is down-regulated at the mRNA level in breast cancer tissues and is up-regulated by steroid hormones in breast and prostate cancer cell lines. This gene may be involved in the pathogenesis and/or progression of certain cancer types and may find applicability as a novel cancer biomarker.     

FISH is more sensitive than Southern analysis at identifying increased levels of cyclin D1 gene amplified in breast cancer cell lines

Cyclin D1 is involved in regulating the transition of G1 to S-phase in the cell cycle through phosphorylation of the retinoblastoma susceptibility product (pRB). Amplification and overexpression of the cyclin D1 gene (CCND1) have been reported in human breast cancers and are suggested to play important roles in the pathogenesis of the disease process. Although cyclin D1 is potentially an important gene, relatively little is known about the distribution of its amplification in breast cancer cell lines. In this study, a cyclin D1 cosmid probe was isolated and used with fluorescence in situ hybridization (FISH) to identify the gene in chromosomal spreads of 12 breast cancer cell lines. Nine cell lines showed increased gene copy levels of cyclin D1, including Five cell lines had more than six copies of cyclin D1 on sister chromatids and four had more than four copies but less than six copies grouped at the chromosome 11 q13 band. Three cell lines had two “normal” chromosome 11 and one and two additional derivative chromosome 11’s with three and four 11q13 sites which lacked amplification of cyclin D1 on any of these sites. Using progesterone receptor (PR) gene as an internal control, a 2.0-fold or greater increase in cyclin D1 gene signals, was observed in five of the ten cell lines by Southern hybridization, the Amplification level of cyclin D1 varied from 2.3 to 19.6-fold. Three cell lines with low amplification of cyclin D1 showed overexpression of the gene by Northern analysis. Our experiments demonstrated that FISH was more sensitive than Southern blot at demonstrating low levels of gene amplification and, additionally, permitted assessment of the distribution of cyclin D1 gene among chromosomes.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Establishment and characterization of three new human breast cancer cell lines derived from Chinese breast cancer tissues

Background  

Breast cancer is a major malignancy affecting females worldwide. It is the most common cause of death from cancer in women. Cell lines are widely used in laboratory research and particularly as in vitro models in cancer research. But we found that the routinely used breast cancer cell lines were mostly derived from Caucasians or African-Americans. There were few standard models to study the pathogenic mechanism at molecular level and cell signaling pathway of breast cancer for Asian patients. It is quite necessary to establish new breast cancer cell lines from xanthoderm to study the pathogenic mechanism and therapeutic methods.     

Specific binding of benzodiazepines to human breast cancer cell lines

Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.     

Chromosome translocations in breast cancer with breakpoints at 8p12

Unbalanced chromosome translocations with breakpoints around 8p12, resulting in loss of distal 8p, are common in carcinomas. We have mapped the 8p12 breakpoints in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1, using YACs and PACs between D8S540 and D8S255 by fluorescence in situ hybridization. All three lines had a breakpoint close to D8S505, proximal to HGL. Each breakpoint was distinct, but all were within 0.5 to 1.5 Mb of each other. The T-47D cell line had a straightforward translocation, but in MDA-MB-361 and ZR-75-1 the translocations were accompanied by local rearrangements of surprising complexity. Small regions of 8p from close to the breakpoint were duplicated or amplified as inserts in the attached chromosome fragment. ZR-75-1 also had retained a separate fragment of about 1 Mb, from the region 1 to 3 Mb telomeric to the common breakpoint, that included HGL. This line also had an interstitial deletion several megabases more centromeric. The data suggest that breakpoints on 8p12 are clustered in a small region and show that translocations breaking there may be accompanied by additional rearrangements.     

The third member of the transforming acidic coiled coil-containing gene family, TACC3, maps in 4p16, close to translocation breakpoints in multiple myeloma, and is upregulated in various cancer cell lines.

We have recently identified a novel gene, TACC1 (transforming acidic coiled coil-containing gene 1), which is located close to FGFR1 within a region amplified in breast cancer on human chromosome 8p11. The coiled coil domain of this gene identified a series of cDNAs in the expressed sequence tag database, which suggested the existence of a family of TACC genes comprising at least three family members. We have now characterized the human and mouse TACC3 cDNAs, and demonstrate that this gene is upregulated in various cancer cell lines, and at Embryonic Day 15 in mice, suggesting that the TACC3 protein is involved in the control of cell growth and differentiation. The TACC3 gene maps telomeric to the FGFR3 gene in 4p16.3, close to a region disrupted by translocation breakpoints associated with multiple myeloma. Thus, TACC1, TACC2, and TACC3 map close to the corresponding FGFR1, FGFR2, and FGFR3 genes. The phylogenetic relationship among the three TACC genes is similar to that of the three FGFR family members. These relationships suggest that the FGFR and TACC genes arose from a physically linked ancestral gene pair. Subsequently, this gene pair has undergone two successive rounds of gene duplication to give rise to the three FGFR/TACC gene pairs on chromosomes 4, 8, and 10.     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

A new Pax gene,Pax-9, maps to mouse Chromosome 12

Members of the Pax gene family have recently been shown to play important roles in mouse embryogenesis. Of eight so far characterized Pax genes, three have been associated with mouse developmental mutants. Here we report the cloning of a new Pax gene, Pax-9. Most of the DNA sequence encoding the highly conserved paired domain has been determined and compared with previously known paired domains. This comparison classifies Pax-9 as a member of the same subgroup as Pax-1/undulated. By analysis of the segregation of a Pax-9 restriction fragment length polymorphism and a large number of simple sequence length polymorphisms in an interspecific C57BL/6 x Mus musculus mollosinus backcross, Pax-9 was mapped close to the D12Nds1 locus on the proximal part of Chromosome (Chr) 12.     

SALL3, a new member of the human spalt-like gene family, maps to 18q23

spalt (sal) of Drosophila melanogaster is an important developmental regulator gene and encodes a zinc finger protein of unusual but characteristic structure. Two human sal-like genes have been isolated so far, SALL1 on chromosome 16q12.1 and SALL2 on chromosome 14q11.1-q12.1. Truncating mutations of SALL1 have been shown to cause Townes-Brocks syndrome and are thought to result in SALL1 haploinsufficiency. Sequence comparison of SALL1 to the related genes Msal in mouse and Xsal-1 in Xenopus laevis suggested that SALL1 was not the human orthologue of Msal and Xsal-1. By database searching and genomic cloning, we isolated an EST and a corresponding human cosmid clone, which contain coding sequence of a human gene highly similar to mouse Msal. This gene, named SALL3, was found to be expressed in different regions of human fetal brain and in different adult human tissues. The chromosomal localization of SALL3 at 18q23 suggests that haploinsufficiency of this gene might contribute to the phenotype of patients with 18q deletion syndrome.     

Comprehensive profiling of 8p11-12 amplification in breast cancer

In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer.     

Development and characterization of new immortalized human breast cell lines

New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media. Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin. They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as models for carcinogenesis studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines

Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.