We have identified an 85 kb BAC clone, 346J21, that carries a cell senescence gene (SEN16), previously mapped to 16q24.3. Transfer and retention of 346J21 in breast cancer cell lines leads to growth ...arrest after 8-10 cell doublings, accompanied by the appearance of characteristic senescent cell morphology and senescence-associated acid beta-galactosidase activity. Loss of transferred BAC results in reversion to the immortal growth phenotype of the parental cancer cell lines. BAC 346J21 restores senescence in the human breast cancer cell lines, MCF.7 and MDA-MB468, and the rat mammary tumor cell line LA7, but not in the human glioblastoma cell line T98G. We postulate that inactivation of both copies of SEN16 is required for the immortalization of breast epithelial cells at an early stage of tumorigenesis. Positional mapping of 346J21 shows that SEN16 is distinct from other candidate tumor suppressor genes reported at 16q24.
Fgf8 (fibroblast growth factor 8) was initially cloned from a mouse mammary tumor cell line derived from the androgen‐dependent Shionogi carcinoma 115. The androgen‐inducible expression of Fgf8 in ...this tumor controls its androgen‐dependent phenotype, thus stimulating interest in this gene as a possible factor in human prostate cancer and other androgen‐sensitive cancers. However, apart from Shionogi carcinoma 115, the androgen inducibility of Fgf8 is controversial. In the present study, having not detected androgen‐inducible expression of Fgf8 in other mouse mammary cell lines or mouse prostate, we examined the Shionogi carcinoma 115‐derived S115 cell line for mouse mammary tumor virus (MMTV) insertions or other nearby DNA rearrangements that might explain the androgen inducibility of Fgf8 in these cells. Southern blotting did not detect MMTV insertions near Fgf8 but did reveal a specific DNA rearrangement 3.7 kb upstream of Fgf8 in S115 cells and in other cells (SC115) independently derived from Shionogi carcinoma 115. Spectral karyotyping of S115 cells and sequencing of the cloned rearrangement junctions indicate that Fgf8 is involved in a t(5;19) translocation. The chromosome 5 sequence joined to Fgf8 is immediately adjacent to Smr2 (submaxillary gland androgen‐regulated protein 2) and includes Muc10 (mucin 10), two genes that we show are testosterone inducible in S115 cells, suggesting that the androgen‐dependent expression of Fgf8 in Shionogi carcinoma 115 and derivative cells results from this translocation. Together, these results suggest that androgen inducibility is not an inherent property of the Fgf8 gene, which has implications regarding this gene's proposed role in the etiology of hormone‐responsive cancers. Published 2005 Wiley‐Liss, Inc.
We previously showed that the human AKNA gene encodes an AT-hook transcription factor that regulates the expression of costimulatory cell surface molecules on lymphocytes. However, AKNA cDNA probes ...hybridize with multiple transcripts, suggesting either the existence of other homologous genes or a complex regulation operating on a single gene. Here we report evidence for the latter, as we find that AKNA is encoded by a single gene that spans a 61-kb locus of 24 exons on the fragile FRA9E region of human chromosome 9q32. This gene gives rise to at least nine distinct transcripts, most of which are expressed in a tissue-specific manner in lymphoid organs. Many of the AKNA transcripts originate from alternative splicing; others appear to derive from differential polyadenylation and promoter usage. The alternative AKNA transcripts are predicted to encode overlapping protein isoforms, some of which (p70 and p100) are readily detectable using a polyclonal anti-AKNA antisera that we generated. We also find that AKNA PEST-dependent cleavage into p50 polypeptides is targeted to mature B cells and appears to be required for CD40 upregulation. The unusual capacity of the AKNA gene to generate multiple transcripts and proteins may reflect its functional diversity, and it may also provide a fail-safe mechanism that preserves AKNA expression.
GC-binding factor 2 (GCF2) is a transcriptional repressor that decreases activity of the epidermal growth factor receptor (EGFR) and other genes. We have mapped the gene for GCF2 by fluorescence in ...situ hybridization (FISH) to chromosome 2q37. Sequence analysis of the GCF2 gene and cDNA showed that the gene consists of eight exons and introns and spans 73 kbp of DNA. Northern blot analysis showed that GCF2 mRNA was differentially expressed in many human tissues and cell lines. GCF2 mRNA was expressed as a 4.2 kb mRNA in most human tissues with the highest expression level in peripheral blood leukocytes and lowest expression in brain and testis. Additional transcripts of 6.6, 2.9 and 2.4 kb were found in some tissues but the only transcript detected in cancer cell lines was 4.2 kb with high levels found in seven Burkitts' lymphoma cell lines. Western blot analysis showed that GCF2 protein is present at high levels in Burkitts' lymphoma and several other cancer cell lines. GCF2 was found in both nuclear and cytoplasmic compartments in cells. Deletion mutants of GCF2 revealed that amino acids 429–528 are required for both DNA binding and repression of the EGFR promoter. Furthermore, GCF2 was able to substantially decrease activator protein 2 (AP2) enhancement of the EGFR promoter. Thus, GCF2 is a transcriptional repressor overexpressed in cancer cell lines with a role in regulating expression of the EGFR.
Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-β-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal ...populations. A receptor complex comprised of TrnR1 (GDNFRα) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are ∼30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2–Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.
Cyclins are essential activators of cyclin-dependent kinases (Cdk) which, in turn, play pivotal roles in controlling transition through cell-cycle checkpoints. Cyclin G2 is a recently discovered ...second member of the G-type cyclins. The two members of the G-type cyclins, cyclin G1 and cyclin G2, share high structural similarity but their function remains to be defined. Here we characterize the structure of the mouse cyclin G2 gene by first cloning and sequencing the full-length mouse cyclin G2 cDNA. The cyclin G2 cDNA was used to isolate the cyclin G2 gene from a BAC library and to establish that the gene was transcribed from eight exons spanning a total of 8604
bp. The cyclin G2 gene was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 5E3.3.–F1.3. This region is syntenic to a region on human chromosome 4. The expression of cyclins G1 and G2 was examined in various tissues, but no correlation between expression patterns of the two genes was observed. However, during hepatic ontogenesis the cyclin G2 expression level decreased with age, whereas cyclin G1 expression increased. Transient expression of cyclin G2-green fluorescent protein (GFP) fusion protein in NIH3T3 cells showed that cyclin G2 is essentially a cytoplasmic protein, in contrast to the largely nuclear localization of cyclin G1. Our data suggest that, despite the close structural similarity between mouse cyclins G1 and G2, these proteins most likely perform distinct functions.
To search for recurrent and specific genomic alterations in human hepatocellular carcinoma (HCC), we examined 18 cell lines by comparative genomic hybridization (CGH), a molecular cytogenetic ...approach that allows positional identification of gains and losses of DNA sequences of the entire tumor genome. We report here a distinct pattern of multiple recurrent DNA copy‐number gains and losses that include alterations frequently seen in other neoplasias as well as changes potentially specific for HCC. The most frequent gains were localized on 1p34.3‐35, 1p33‐34.1, 1q21‐23, 1q31‐32, 6p11‐12, 7p21, 7q11.2, 8q24.1‐24.2, 11q11‐13, 12q11‐13, 12q23, 17q11.2‐21, 17q23‐24, and 20p11.1‐q13.2. Recurrent losses were mapped on 3p12‐14, 3q25, 4p12‐14, 4q13‐34, 5q21, 6q25‐26, 8p11.2‐23, 9p12‐24, 11q23‐24, 13q12‐33, 14q12‐13, 15q25‐26, 18q11.2‐22.2, and 21q21‐22. Seventeen genomic imbalances are novel in HCC, thus extending significantly the map of genetic changes and providing a starting point for the isolation of new genes relevant in pathogenesis of liver neoplasia, as well as providing molecular probes for both diagnosis and monitoring treatment of the disease
DLC1 (Deleted in Liver Cancer 1) gene encodes a RhoGTPase-activating protein (RhoGAP), which exerts most of its tumor suppressor functions through suppression of small Rho GTPases proteins RhoA, ...RhoB, RhoC and to some degree Cdc42, but not Rac. RhoGTPases are implicated in NF-kappaB activation in highly invasive prostate carcinoma (PCA), with consequences on cell proliferation, survival and metastatic capacity. Here we demonstrate that DLC1 transduction in two androgen-independent (AI) and highly metastatic PCA cell lines negatively regulates NF-kappaB activity in a GAP- and alpha-catenin-dependent manner. Expressed DLC1 protein suppresses the phosphorylation of NF-kappaB inhibitor, IkappaBalpha, causes its relocation from membrane ruffles into cytoplasm and attenuates its ubiquitination and subsequent degradation. DLC1-mediated NF-kB suppression and its effects are comparable to NF-kappaB inhibition using either shRNA knockdown or peptide inhibitor. Expression of transduced DLC1 suppressed the expression of NF-kappaB mediated genes. Such effects were found to be reliant on presence of calcium, indicating that the observed modifications are dependent on, and enabled by DLC-mediated stabilization of adherens junctions. These results expand the multitude of DLC1 interactions with other genes that modulate its oncosuppressive function, and may have potential therapeutic implications.
We previously identified KEPI as a morphine-regulated gene using subtractive hybridization and differential display PCR. Upon phosphorylation by protein kinase C, KEPI becomes a powerful inhibitor of ...protein phosphatase 1. To gain insights into KEPI functions, we created KEPI knockout (KO) mice on mixed 129S6 × C57BL/6 genetic backgrounds. KEPI maps onto mouse chromosome 10 close to the locus that contains the μ-opioid receptor (Oprm1) and provides a major quantitative trait locus for morphine effects. Analysis of single nucleotide polymorphisms in and near the Oprm1 locus identified a doubly-recombinant mouse with C57BL/6 markers within 1 Mb on either side of the KEPI deletion. This strategy minimized the amount of 129S6 DNA surrounding the transgene and documented the C57BL/6 origin of the Oprm1 gene in this founder and its offspring. Recombinant KEPIKO mice displayed a) normal analgesic responses and normal locomotion after initial morphine treatments, b) accelerated development of tolerance to analgesic effects of morphine, c) elevated activity of protein phosphatase 1 in thalamus, d) attenuated morphine reward as assessed by conditioned place preference. These data support roles for KEPI action in adaptive responses to repeated administration of morphine that include analgesic tolerance and drug reward.
Background Two neoplastic Kaposi's sarcoma (KS) cell lines, KS Y-1 (derived from a patient with KS associated with acquired immunodeficiency syndrome) and KS SLK (derived from an immunosuppressed ...patient with a renal transplant and KS or iatrogenic KS), have been shown to have abnormal chromosome constitution and to require no exogenous growth factors. They produce malignant tumors in immunodeficient mice. In contrast, all other cell cultures prepared in the past from KS specimens have been shown to have normal diploid characteristics, are hyperplastic, and depend on cytokines for growth, but they do not produce malignant tumors in immunodeficient mice. Purpose We investigated whether the chromosomal changes that occurred in these KS cell lines were random or nonrandom and if such changes contribute to the pathogenesis of KS. Methods We used the conventional G-banding technique and fluorescence in situ hybridization to identify structural and numerical chromosomal changes in the KS cell lines. Results We demonstrated that both cell lines are aneuploid and have some additional features in common, i.e., loss of copies of chromosomes 14 and 21 and nonrandom translocations and deletions in the short arm of chromosome 3 at region 3p14. These KS cell lines also exhibit loss of heterozygosity of loci at region 3p14-ter. Conclusion This is the first time nonrandom chromosomal alterations have been described in KS neoplastic cells. On the basis of information available on other cancers, the chromosome 3 alterations observed here can be expected to contribute to the neoplastic process in KS. Implications Future research should focus on the identification of cytogenetic markers, thus facilitating generation of specific molecular probes for detecting neoplastic cells early in the disease process. J Natl Cancer Inst 1996;88:450–5
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