XIPHOPHORUS ENZYME GENE EXPRESSION: TISSUE SPECIFICITY, GENETIC CONTROL, AND STABILITY IN CULTURED CELLS

Five permanent cell lines were developed from Xiphophorus maculatus, X. helleri, and their hybrids using three tissue sources, including adults and embryos of different stages. To evaluate cell line gene expression for retention of either tissue-of-origin-specific or ontogenetic stage-specific characters, the activity distribution of 44 enzyme loci was determined in 11 X. maculatus tissues, and the developmental genetics of 17 enzyme loci was charted in X. helleri and in helleri x maculatus hybrids using starch gel electrophoresis. In the process, eight new loci were discovered and characterized for Xiphophorus.^ No Xiphophorus cell line showed retention of tissue-of-origin-specific or ontogenetic stage-specific enzyme gene expressional traits. Instead, gene expression was similar among the cell lines. One enzyme, adenosine deaminase (ADA) was an exception. Two adult-origin cell lines expressed ADA, whereas, three cell lines derived independently from embryos did not. ADA('-) expression of Xiphophorus embryo-derived cell lines may represent retention of an embryonic gene expressional trait. In one cell line (T(,3)) derived from 13 pooled interspecific hybrid (F(,2)) embryos, shifts with time were observed at enzyme loci polymorphic between the two species. This suggested shifts in ratios of cells of different genotypes in the population rather than unstable gene expression in one dominant cell type.^ Verification of this hypothesis was attempted by cloning the culture--seeking clones having different genetic signatures. The large number of loci electrophoretically polymorphic between the two species and whose alleles segregated independently into the 13 progeny from which this culture originated almost guaranteed the presence of different genetic signatures (lineages) in T(,3).^ Seven lineages of cells were found within T(,3), each expressing genotypes at some loci not characteristic of the expression of the culture-as-a-whole, supporting the hypothesis tested. Quantitative studies of ADA expression in the whole culture (ADA('-)) and in clones of these seven lineages suggested the predominance in T(,3) of ADA deficient cell lineages, although moderate to high ADA output clones also occurred. Thus, T(,3) has the potential to shift phenotypes from ADA('-) to ADA('+) by simply changing proportions of its constituent cell types, demonstrating that such shifts can occur in any cell culture containing cells of mixed expressional characteristics.^

The role of post-translation regulation of Twist expression in the tumor progression of squamous cell carcinoma of head and neck

Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://scansite.mit.edu/motifscan_seq.phtml ), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addition, mutation of the putative CK2 phosphorylation site (S18/S20A) in TWIST decreased the amount of phospho-ATP incorporated by TWIST in an in vitro kinase assay, and altered TWIST stability. In Boyd chamber migration assay and wound-healing assay, the CK2 inhibitor, DMAT, was found to decrease the motility of IL-6 stimulated SCCHN cells and over expression of either a wild-type or the hyperphosphorylated mimicking mutant S18/20D –Twist rather than the hypo-phosphorylated mimicking mutant S18/20A-Twist can promote SCCHN cell motility.To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway.

14-3-3SIGMA NEGATIVELY REGULATES THE STABILITY AND SUBCELLULAR LOCALIZATION OF COP1

Mammalian constitutive photomorphogenic 1 (COP1), a p53 E3 ubiquitin ligase, is a key negative regulator for p53. DNA damage leads to the translocation of COP1 to the cytoplasm, but the underlying mechanism remains unknown. We discovered that 14-3-3σ controlled COP1 subcellular localization and protein stability. Investigation of the underlying mechanism suggested that, upon DNA damage, 14-3-3σ bound to phosphorylated COP1 at S387, resulting in COP1 translocation to the cytoplasm and cytoplasmic COP1 ubiquitination and proteasomal degradation. 14-3-3σ targeted COP1 for degradation to prevent COP1-mediated p53 degradation, p53 ubiquitination, and p53 transcription repression. COP1 expression promoted cell proliferation, cell transformation, and tumor progression, attesting to its role in cancer promotion. 14-3-3σ negatively regulated COP1 function and prevented tumor growth in cancer xenografts. COP1 protein levels were inversely correlated with 14-3-3σ protein levels in human breast and pancreatic cancer specimens. Together, these results define a novel, detailed mechanism for the posttranslational regulation of COP1 upon DNA damage and provide a mechanistic explanation of the correlation of COP1 overexpression with 14-3-3σ downregulation during tumorigenesis.

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

Analysis of mutations in $\beta$-tubulin genes affecting tubulin stability and assembly

Cmd4 is a colcemid-sensitive CHO cell line that is temperature sensitive for growth and expresses an altered $\beta$-tubulin, $\beta\sb1$. One revertant of this cell line, D2, exhibits a further alteration in $\beta\sb1$ resulting in an acidic shift in its isoelectric point and a decrease in its molecular weight to 40 kD, as measured by two dimensional gel electrophoresis. This $\beta$-tubulin variant has been shown to be assembly-defective and unstable. Characterization of the mutant $\beta\sb1$ in D2 by high pressure liquid chromatography (HPLC) revealed the loss of methionine containing tryptic peptides 7,8,9, and 10. Southern analysis of the genomic DNA digested with several different restriction enzymes resulted in the appearance of new restriction fragments 250 base pairs shorter than the corresponding fragments from the wild-type $\beta\sb1$-tubulin gene. Northern analysis on mRNA from D2 revealed two new message products that also differed by 250 bases from the corresponding wild type $\beta$-tubulin transcripts. To precisely define the region of the alteration, cloning and sequencing of the mutant and wild type genomic $\beta$-tubulin genes were conducted. A size-selected EcoRI genomic library was prepared using the Stratagene lambda Zap II phage cloning system. Using subclones of CHO $\beta$-tubulin cDNA as probes, a 2.5 kb wild type clone and a 2.3 kb mutant clone were identified from this library. Each of these was shown to contain a portion of the gene extending from intron 3 through the end of the coding sequence in exon 4 and into the 3$\sp\prime$ untranslated region on the basis of alignment with the published human $\beta$-tubulin sequence. Sequencing of the mutant 2.3 kb clone revealed that the mutation is due to a 246 base pair internal deletion in exon 4 (base pair 756-1001) that encodes amino acids 253-334. This deletion results in the loss of a putative binding site for GTP which could potentially explain the phenotype of this mutant $\beta$-tubulin. Also sequence comparison of the 3$\sp\prime$ untranslated region between different species revealed the conservation of 200 base pairs with 78% homology. It is proposed that this region could play an important role in the regulation of $\beta$-tubulin gene expression. ^

Regulation of the expression of {\it mutS, mutL}, and {\it mutH\/} mismatch repair genes in {\it Escherichia coli\/} K-12

Post-replication DNA mismatch repair plays crucial roles in mutation avoidance and maintenance of chromosome stability in both prokaryotes and eukaryotes. In humans, deficiency in this repair system leads to a predisposition for certain cancers. The biochemistry of this repair system has been best studied in a model bacterium Escherichia coli. In this thesis, regulation of expression of mutS, mutL and mutH genes, whose products mediate methyl-directed mismatch (MDM) repair in E. coli, is investigated. One-step affinity purification schemes were developed to purify E. coli MutS, MutL and MutH proteins fused to a His-6-affinity tag. His-6-MutS exhibited the same mismatch binding activity and specificity as the native MutS protein. Purified His-6-MutS, -MutL and -MutH proteins were used to develop quantitative Western blotting assays for amounts of MutS, MuL and MutH proteins under various conditions. It was found that the three proteins were present in relatively low amounts in exponentially growing cells and MutS and MutH were diminished in stationary-phase cells. Further studies indicated that the drop in the amounts of MutS and MutH proteins in stationary-phase cells was mediated through RpoS, a key global regulator of stationary-phase transition. In both exponential- and stationary-phase cells, MutS amount was also negatively regulated by the Hfq (HF-I) global regulator, which is required for RpoS translation, through an RpoS-independent mechanism. $\beta$-galactosidase assays of mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally, and RNase T2 protection assays revealed that Hfq destabilizes mutS transcripts in exponentially growing cells. To study the relation between regulation of MDM repair and mutagenesis, amounts of MutS, MutL and MutH were measured in starved cells undergoing adaptive mutagenesis. It was found that MutS amount dropped drastically, MutH amount dropped slightly, whereas MutL amount remained essentially constant in starved cells. Overexpression of MutL did not reverse the drop in the amounts of MutS or MutH protein. These results ruled out several explanations for a phenomenon in which overexpression of MutL, but not MutS, reversed adaptive mutagenesis. The findings further suggested that functional MutL is limiting during adaptive mutagenesis. The implications of regulation of the MDM repair are discussed in the context of mutagenesis, pathogenesis and tumorigenesis. ^

A TEMPERATURE-SENSITIVE DEFECT IN THE EXPRESSION OF A SARCOMA VIRUS TRANSFORMING GENE

The viral proteins synthesized by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in a function required for the maintenance of the transformed state (ts110) were examined. Normal rat kidney cells (NRK) were infected with the ts110 virus and a non-virus-producing cell clone, termed 6m2, was isolated. This cell clone had a malignant phenotype at 33(DEGREES), the permissive temperature, but changed to a normal phenotype at 39(DEGREES).^ Two viral proteins were detected in 6m2 cells. A 58,000 dalton protein (P58) was detected at both 33(DEGREES) and 39(DEGREES) and contained only core protein (gag) coded sequences. An 85,000 dalton protein (P85) was detected only at 33(DEGREES) and contained sequences of viral core proteins p15, pp12, and part of p30 as well as protein sequences attributed by peptide mapping to P23 and P38, two candidate viral mouse src (v-mos) gene products. These results provide good evidence that P85 is a gag-mos polyprotein. As expected for a functional mos-gene product, P85 synthesis preceded parameters characteristic of the transformed state, including changes in cell morphology, in the cytoplasmic microtubule complex (CMTC) and in the rate of hexose uptake.^ Other studies were conducted to ascertain the defect which prohibited the synthesis of P85 at 39(DEGREES), the non-permissive temperature. When 6m2 cells were treated with actinomycin D at 39(DEGREES) and shifted to 33(DEGREES), the cells were unable to synthesize P85, but P58 continued to be made. P85 mRNA, active at 33(DEGREES), continued to be translated for two to three hours after shifting to 39(DEGREES) as judged by pulse-labeling experiments. Virus harvested at 33(DEGREES) from ts110 MuSV producer cells packaged both P85 and P58 coding RNAs while virus harvested at 39(DEGREES) was deficient in the amount of P85 coding RNA. Agarose gel electrophoresis of 6m2 cellular RNA showed that RNA harvested at 33(DEGREES) contained the 4.0 and 3.5 kb RNAs. Similar experiments on cells maintained at 39(DEGREES) have detected only the 4.0 kb RNA, suggesting that the 3.5 kb RNA codes for P85. The defect appeared to be in the long term stability of the P85 coding RNA at 39(DEGREES), since, in shift-up experiments (33(DEGREES) (--->) 39(DEGREES)), P85 was translated for only three hours at 39(DEGREES), while P58 was synthesized for at least eight hours. However, at 33(DEGREES) in the presence of actinomycin D, the ratio of P85 and P58 synthesis at hourly intervals was similar throughout a 12 hour period. ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^