PTTG1 Levels Are Predictive of Saracatinib Sensitivity in Ovarian Cancer Cell Lines.

Src kinase is recognized as a key target for molecular cancer therapy. However, methods to efficiently select patients responsive to Src inhibitors are lacking. We explored the sensitivity of ovarian cancer cell lines to the Src kinase inhibitor saracatinib to identify predictive markers of drug sensitivity using gene microarrays. Pituitary tumor transforming gene 1 (PTTG1) was selected as a potential biomarker as mRNA levels were correlated with saracatinib resistance, as well as higher PTTG1 protein expression. PTTG1 expression was correlated with proliferation, cell division, and mitosis in ovarian cancer tissues data sets. In sensitive cell lines, saracatinib treatment decreased PTTG1 and fibroblast growth factor 2 (FGF2) protein levels. Downregulating PTTG1 by siRNAs increased saracatinib sensitivity in two resistant cell lines. Our results indicate PTTG1 may be a valuable biomarker in ovarian cancer to predict sensitivity to saracatinib, and could form the basis of a targeted prospective saracatinib trial for ovarian cancer.

Development of a native plasmid as a cloning vector in Clavibacter xyli subsp. cynodontis.

A 50-kilobase (kb) cryptic plasmid was found in three geographic isolates of Clavibacter xyli subsp. cynodontis (Cxc). The DNA region essential for replication of the plasmid was cloned in an Escherichia coli vector. The resulting vector, which functions as a shuttle vector between E. coli and Cxc, was characterized further with respect to its stability and copy number.

Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis.

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.

Combining thermostable mutations increases the stability of lambda repressor.

We have combined three mutations previously shown to stabilize lambda repressor against thermal denaturation. Two of these mutations are in helix 3, where Gly-46 and Gly-48 have been replaced by alanines [Hecht, M. H., et al. (1986) Proteins: Struct., Funct., Genet. 1, 43-46]. The other mutation, which replaces Tyr-88 with cysteine, allows the protein to form an intersubunit disulfide bond [Sauer, R. T., et al. (1986) Biochemistry 25, 5992-5998]. Calorimetric measurements show that the two alanine substitutions stabilize repressor by about 8 degrees C, that the disulfide bond stabilizes repressor by about 8 degrees C, and that the triple mutant is 16 degrees C more stable than wild-type repressor.

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.

Structure of the murine serum amyloid A gene family. Gene conversion.

Serum amyloid A (SAA) is an apolipoprotein produced by the liver in response to inflammation; the levels of SAA mRNA and SAA protein increase at least 500-fold within 24 h. We have obtained clones of all three genes and pseudogene that make up the murine SAA gene family. Two of the genes have 96% sequence homology over their entire length, including introns and flanking sequences 288 base pairs (bp) 5' and 443 bp 3' to the genes: an overall length of 3215 bp. The sharp boundaries between homologous and nonhomologous sequences and the absence of interspersed repeated sequences there suggest that conversion has occurred between these two genes. The homologous regions are bounded by short inverted repeats containing alternating purine and pyrimidine residues, as described for other gene conversion units. The third SAA gene has evolved separately, although all are closely linked on chromosome 7. Comparison of the upstream regions of the SAA genes with those of the rat fibrinogen genes, whose expression is also induced by inflammation, reveals sequences common to all six genes which are very improbable on a random basis.

Transcriptional regulation of serum amyloid A gene expression.

Serum amyloid A (SAA) is a plasma apolipoprotein produced by the liver in response to inflammatory stimuli. The murine SAA gene family is made up of three genes, SAA1, SAA2, and SAA3, plus a pseudogene. The SAA1 and SAA2 genes are highly homologous while the SAA3 gene has diverged substantially from the other two genes. Using small fragments from the cloned genes, we have analyzed the expression of each gene in the SAA family. Within 12 h after endotoxin administration, total liver SAA mRNA increases by 2000-fold, reaching approximately 20,000 transcripts/cell. Each gene accounts for approximately one-third of total SAA mRNA transcripts at this time. The increase is specific, since the levels of the mRNAs encoding albumin and apolipoprotein A-I in liver decrease 2-fold by 24 h. This correlates with a 2-fold decrease of the serum concentrations of these two proteins as well as their in vitro protein synthesis in primary hepatocytes. SAA1+2 mRNAs maintain their maximum levels until 36 h after lipopolysaccharide administration, while SAA3 mRNA is degraded to 20% its maximal level. As assayed by in vitro transcription in isolated hepatocyte nuclei, total SAA gene transcription increases at least 300-fold during the inflammatory response. The transcription rates of the individual SAA genes are similar during the initial stages of this response, reaching peak levels at 3 h. A comparison of the rates of SAA gene transcription and SAA mRNA accumulation suggests that SAA mRNA levels are regulated during the acute phase response by increased transcription and mRNA stabilization.

The sequence and structure of a new serum amyloid A gene.

The acute phase response is characterized by changes in the serum concentrations of many proteins. A 1000-fold increase in the concentration of serum amyloid A (SAA) protein occurs within 24 hours of LPS injection in the mouse. We have isolated a cDNA clone and its corresponding genomic phage for a third, previously unreported SAA protein. The sequence of the cDNA, the gene's exons and neighboring DNA are presented along with the mapping evidence supporting the gene structure.

Regulation of synthesis of amyloid A-related protein.

The concentration of serum amyloid A polypeptide (SAAL) increases greatly during the acute phase responses to infection or inflammation. We find that SAAL synthesis comprises 2.5% of murine hepatic protein synthesis after lipopolysaccharide (LPS) administration, but much less in normal liver. SAAL messenger RNA (mRNA) in liver increases at least 500-fold above the normal level. A recombinant plasmid homologous to SAAL mRNA has been isolated, as has most of the mouse genome DNA encoding the plasmid's nucleotide sequence. This gene is transcribed into RNA much more frequently after LPS administration than it is in normal liver. In a number of other mammalian genes, cytosine methylation is inversely related to the rate of transcription. Methylation of CCGG sequences in hepatic DNA homologous to the recombinant plasmid has been examined. Little or no change is found after LPS administration. This suggests that other factors are responsible for the increase in SAAL mRNA in the acute phase response.

Induction of hepatic synthesis of serum amyloid A protein and actin.

Major changes in the mRNA population of murine liver occur after administration of bacterial lipopolysaccharide, an agent that causes increases in the concentrations of acute-phase serum proteins. The mRNA for one of these, serum amyloid A, is increased at least 500-fold compared to the normal level. It becomes one of the most abundant hepatic mRNAs, and serum amyloid A synthesis comprises about 2.5% of total hepatic protein synthesis in the acute-phase response. Its synthesis is tissue-specific in that amyloid A mRNA was not detected in the kidney, an important site of amyloid fibril accumulation. The protein synthesized in largest amount by acute-phase liver tissue in culture is cytoplasmic actin. Its relative rate of synthesis is increased about 5-fold compared to the normal tissue; that of serum albumin is decreased to about one-third of its normal rate. The concentration of mRNA for serum albumin is decreased by a similar amount. Starting with induced liver RNA, we have constructed a recombinant plasmid containing most of the DNA sequence encoding the serum amyloid A polypeptide.