KRAS mutational subtype and copy number predict in vitro response of human pancreatic cancer cell lines to MEK inhibition.

BACKGROUND: To study the molecular mechanism regulating sensitivity to MEK inhibition in pancreatic cancer cell lines. METHODS: A growth inhibition assay determined sensitivity to MEK162 in a panel of 29 pancreatic cancer cell lines. For the same panel, KRAS mutational status and copy-number variation (CNV) was determine using PCR, array CGH and FISH. Two sensitive and two resistant cell lines were further interrogated for difference in baseline and MEK162-induced gene expression, as well as signal transduction using microarray and western blotting. Cell cycle and apoptosis analysis was measured by flow cytometry. RESULTS: We report a strong correlation between both specific KRAS mutational subtype and CNV, and sensitivity to MEK inhibition. Cell lines with a KRAS (V12) mutation and KRAS gains or loss (n=7) are ∼10 times more resistant than those having neither a KRAS (V12) mutation nor KRAS CNV (n=14). Significant differences in baseline and MEK162-induced gene expression exist between the sensitive and resistant lines, especially in genes involved in RAS, EGF receptor and PI3K pathways. This was further supported by difference in signal transduction. MEK 162 blocked ERK1/2, as well as inhibited PI3K and S6 and increased p27KIP1 levels in the sensitive lines. CONCLUSIONS: Given the potency of MEK162, it may be a promising new therapy for patients with pancreatic cancer and KRAS mutational subtypes, and CNV may serve as important biomarkers for selecting patients that benefit from MEK-targeting based on these preclinical data.

FOXA1 represses the molecular phenotype of basal breast cancer cells.

Breast cancer is a heterogeneous disease that comprises multiple subtypes. Luminal subtype tumors confer a more favorable patient prognosis, which is, in part, attributed to estrogen receptor (ER)-α positivity and antihormone responsiveness. Expression of the forkhead box transcription factor, FOXA1, similarly correlates with the luminal subtype and patient survival, but is also present in a subset of ER-negative tumors. FOXA1 is also consistently expressed in luminal breast cancer cell lines even in the absence of ER. In contrast, breast cancer cell lines representing the basal subtype do not express FOXA1. To delineate an ER-independent role for FOXA1 in maintaining the luminal phenotype, and hence a more favorable prognosis, we performed expression microarray analyses on FOXA1-positive and ER-positive (MCF7, T47D), or FOXA1-positive and ER-negative (MDA-MB-453, SKBR3) luminal cell lines in the presence or absence of transient FOXA1 silencing. This resulted in three FOXA1 transcriptomes: (1) a luminal signature (consistent across cell lines), (2) an ER-positive signature (restricted to MCF7 and T47D) and (3) an ER-negative signature (restricted to MDA-MB-453 and SKBR3). Gene set enrichment analyses revealed FOXA1 silencing causes a partial transcriptome shift from luminal to basal gene expression signatures. FOXA1 binds to a subset of both luminal and basal genes within luminal breast cancer cells, and loss of FOXA1 increases enhancer RNA transcription for a representative basal gene (CD58). These data suggest FOXA1 directly represses a subset of basal signature genes. Functionally, FOXA1 silencing increases migration and invasion of luminal cancer cells, both of which are characteristics of basal subtype cells. We conclude FOXA1 controls plasticity between basal and luminal breast cancer cells, not only by inducing luminal genes but also by repressing the basal phenotype, and thus aggressiveness. Although it has been proposed that FOXA1-targeting agents may be useful for treating luminal tumors, these data suggest that this approach may promote transitions toward more aggressive cancers.

Activity of the multikinase inhibitor dasatinib against ovarian cancer cells.

BACKGROUND: Here, we explore the therapeutic potential of dasatinib, a small-molecule inhibitor that targets multiple cytosolic and membrane-bound tyrosine kinases, including members of the Src kinase family, EphA2, and focal adhesion kinase for the treatment of ovarian cancer. METHODS: We examined the effects of dasatinib on proliferation, invasion, apoptosis, cell-cycle arrest, and kinase activity using a panel of 34 established human ovarian cancer cell lines. Molecular markers for response prediction were studied using gene expression profiling. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions with chemotherapeutic drugs. RESULTS: Concentration-dependent anti-proliferative effects of dasatinib were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC(50) values (IC(50) range: 0.001-11.3 micromol l(-1)). Dasatinib significantly inhibited invasion, and induced cell apoptosis, but less cell-cycle arrest. At a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for dasatinib plus carboplatin (mean CI values, range: 0.73-1.11) or paclitaxel (mean CI values, range: 0.76-1.05). In this study, 24 out of 34 (71%) representative ovarian cancer cell lines were highly sensitive to dasatinib, compared with only 8 out of 39 (21%) representative breast cancer cell lines previously reported. Cell lines with high expression of Yes, Lyn, Eph2A, caveolin-1 and 2, moesin, annexin-1, and uPA were particularly sensitive to dasatinib. CONCLUSIONS: These data provide a clear biological rationale to test dasatinib as a single agent or in combination with chemotherapy in patients with ovarian cancer.

Activity of lapatinib a novel HER2 and EGFR dual kinase inhibitor in human endometrial cancer cells.

In this study, we explore the therapeutic potential of lapatinib a selective inhibitor of both the EGFR and HER2 tyrosine kinases for the treatment of endometrial cancer. The effect of lapatinib on tumour cell growth and receptor activation was studied in a panel of human endometrial cancer cell lines. Candidate molecular markers predicting sensitivity were assessed by baseline gene expression profiling, ELISA, and western blot analyses. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions between chemotherapeutic drugs and lapatinib. Concentration-dependent anti-proliferative effects of lapatinib were seen in all endometrial cancer cell lines tested, but varied significantly between individual cell lines (IC(50) range: 0.052-10.9 micromol). HER2 overexpression or increased expression of EGFR was significantly associated with in vitro sensitivity (P=0.024 or 0.011, respectively). Lapatinib exerts growth inhibition in a PTEN-independent manner. Sensitive cell lines also exhibited increased expression of EGFR ligands or HER3. In contrast, lapatinib-resistant cell lines exhibited high androgen receptor (AR) levels or epithelial-to-mesenchymal transition (post-EMT) features. In endometrial cancer cells, at a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for lapatinib plus carboplatin, paclitaxel, docetaxel, and doxorubicin. These observations provide a clear biologic rational to test lapatinib as a single agent or in combination with chemotherapy in endometrial cancer with HER2 overexpression. Expression of EGFR, its ligands, HER3, AR, and post-EMT markers warrant further evaluation to help define patients with HER2-nonoverexpressing endometrial cancer most likely to benefit from lapatinib.

A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers.

Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. We have identified two human homologues of these genes, termed aurora1 and aurora2, that encode cell-cycle-regulated serine/threonine kinases. Here we demonstrate that the aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies. We propose that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy.

Extent of heterogeneity in mitochondrial DNA of sub-Saharan African populations.

Variation in the mitochondrial DNA (mtDNA) control region as detected by sequence-specific oligonucleotide (SSO) probes is described for 381 individuals from nine sub-Saharan African populations. Population diversity estimates for SSO types ranged from 0.23 to 0.97, while 102 SSO types were detected, none of these types was shared by more than four populations. Eighteen types occurred in > or = 10% of individuals in some populations; of these, 11 were population-specific. One type occurred in 15% of the total sample, but was shared among only three populations. African SSO types were characterized by high frequencies of blank variants, indicating that there was additional variation present at the nucleotide sequence level in regions where SSO probes hybridize. Analyses of molecular variance (AMOVA) incorporating genetic distances between SSO types showed that 30% of the total variation was due to differences among populations, indicating that there is statistically significant heterogeneity (p < 0.001). An AMOVA on mtDNA control region nucleotide sequence data from 12 populations showed that including all additional variation present at the sequence level increased the variance due to population subdivision to 34% (p < 0.001). Overall, when considering both the low diversity within some populations and high heterogeneity among populations, SSO typing of mtDNA may not be a desirable forensic DNA typing method for continental African populations. Further mtDNA sampling of African-derived populations of North America should be carried out to determine how much of the continental African mtDNA variation is of forensic significance. However, the existence of extensive mtDNA control region nucleotide sequence variation in African populations means that control region sequencing is still appropriate in forensic cases requiring mtDNA analysis.

DNA sequencing using a four-color confocal fluorescence capillary array scanner.

The design, construction and operation of a four-color capillary array electrophoresis scanner are presented. The use of sensitive energy transfer primers facilitates four-color detection of the DNA sequencing fragments following excitation at a single laser wavelength (488 nm). This scanner collects fluorescence data from up to 25 capillaries in parallel. The resulting four-color image files are automatically reduced to four-color line plots, and a base-calling program (Sax) is used to call the sequence. The performance of this system for DNA sequencing is demonstrated by examining twelve different motifs of the hypervariable region I of human mitochondrial (mt) DNA obtained from a Sierra Leone population.

Mitochondrial D-loop "signatures" produced by low-stringency single specific primer PCR constitute a simple comparative human identity test.

We have developed a technique called "LSSP-PCR" (low-stringency single specific primer PCR) that detects single or multiple mutations in DNA. A purified DNA fragment is submitted to PCR by using a single primer specific for one of the extremities of the fragment, under conditions of very low stringency. The primer hybridizes specifically to its complementary extremity and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner. A complex set of reaction products is thus created that, when separated by electrophoresis, constitutes a unique "gene signature." We here report the application of LSSP-PCR to the detection of sequence variation in the control (D-loop) region of human mtDNA, which is known to differ significantly between unrelated individuals. We prepared human DNA samples from blood and amplified a 1024-bp portion of the mtDNA control region, using primers L15996 and H408. The amplified mtDNA fragments were then reamplified under LSSP-PCR conditions by using L15996 or H408 as drivers to produce complex signatures that always differed between unrelated individuals and yet were highly reproducible. In contrast, all mother-child pairs tested were identical, as expected from the matrilineal inheritance of mtDNA. Thus, the use of LSSP-PCR to produce D-loop signatures constitutes a powerful new technique for mtDNA-based comparative identity testing.

Genetic variation among the Mapuche Indians from the Patagonian region of Argentina: mitochondrial DNA sequence variation and allele frequencies of several nuclear genes.

DNA samples from 60 Mapuche Indians, representing 39 maternal lineages, were genetically characterized for (1) nucleotide sequences of the mtDNA control region; (2) presence or absence of a nine base duplication in mtDNA region V; (3) HLA loci DRB1 and DQA1; (4) variation at three nuclear genes with short tandem repeats; and (5) variation at the polymorphic marker D2S44. The genetic profile of the Mapuche population was compared to other Amerinds and to worldwide populations. Two highly polymorphic portions of the mtDNA control region, comprising 650 nucleotides, were amplified by the polymerase chain reaction (PCR) and directly sequenced. The 39 maternal lineages were defined by two or three generation families identified by the Mapuches. These 39 lineages included 19 different mtDNA sequences that could be grouped into four classes. The same classes of sequences appear in other Amerinds from North, Central, and South American populations separated by thousands of miles, suggesting that the origin of the mtDNA patterns predates the migration to the Americas. The mtDNA sequence similarity between Amerind populations suggests that the migration throughout the Americas occurred rapidly relative to the mtDNA mutation rate. HLA DRB1 alleles 1602 and 1402 were frequent among the Mapuches. These alleles also occur at high frequency among other Amerinds in North and South America, but not among Spanish, Chinese or African-American populations. The high frequency of these alleles throughout the Americas, and their specificity to the Americas, supports the hypothesis that Mapuches and other Amerind groups are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)

Identifying individuals by sequencing mitochondrial DNA from teeth.

Mitochondrial DNA (mtDNA) was extracted from teeth stored from 3 months to 20 years, including teeth from the semi-skeletonized remains of a murder victim which had been buried for 10 months. Tooth donors and/or their maternal relatives provided blood or buccal cells, from which mtDNA was also extracted. Enzymatic amplification and direct sequencing of roughly 650 nucleotides from two highly polymorphic regions of mtDNA yielded identical sequences for each comparison of tooth and fresh DNA. Our results suggest that teeth provide an excellent source for high molecular weight mtDNA that can be valuable for extending the time in which decomposed human remains can be genetically identified.

Intergenic suppression of stage II sporulation defects by a mutation in the major vegetative sigma factor gene (rpoD) of Bacillus subtilis.

The Bacillus subtilis intergenic suppressor mutations crsA and rvtA, previously shown to restore sporulation competence to a variety of strains containing stage 0 sporulation defects, also suppress lesions in the stage II sporulation genes spoIIF, spoIIN and spoIIJ. They do not rescue sporulation in other stage II through stage V sporulation mutations. Cells containing spoIIN, spoIIF96 and spoIIJ::Tn917 mutations fail to transcribe spoIID, a late stage II gene. Introduction of crsA47 into spoIINts279, spoIIF96, or spoIIJ::Tn917 mutant backgrounds circumvents the need for the spoIIF, IIN, and IIJ products, restoring both expression of spoIID, and sporulation competence.

Roles of rpoD, spoIIF, spoIIJ, spoIIN, and sin in regulation of Bacillus subtilis stage II sporulation-specific transcription.

Bacillus subtilis strains containing defects in the sporulation gene spoIIF (kinA), spoIIJ (kinA), or spoIIN (ftsA) cannot transcribe the sigma E-dependent gene spoIID. Results presented here and by other workers demonstrate that the spoIIF, spoIIJ, and spoIIN gene products control spoIID transcription indirectly by coordinating the induction of the spoIIGAB, spoIIE, and spoIIAC operons, which are required for sigma E synthesis and processing. Sporulation competence and spoIIGAB, spoIIE, and spoIIAC transcription were restored in spoIIF, spoIIJ, and spoIIN mutants by introduction of crsA47, a mutation in the major vegetative sigma factor sigma A. crsA mutations are known to restore sporulation in certain spo0 mutants. crsA suppression of kinA and ftsA mutations was achieved through inhibition of the transcription of sin, a gene involved in the selection between several post-exponential-phase cell states. A deletion of sin restored sporulation competence in spoIIF, spoIIJ, or spoIIN mutant strains. A sin deletion was also able to restore sporulation competence in the crsA suppressible stage 0 mutant spo0K141.

Suppression of defective-sporulation phenotypes by mutations in transcription factor genes of Bacillus subtilis.

Mutations in the Bacillus subtilis major RNA polymerase sigma factor gene (rpoD/crsA47) and a sensory receiver gene (spoOA/rvtA11) are potent intergenic suppressors of several stage 0 sporulation mutations (spoOB, OE, OF & OK). We show here that these suppressors also rescue temperature-sensitive sporulation phenotypes (Spots) caused by mutations in RNA polymerase, ribosomal protein, and protein synthesis elongation factor EF-G genes. The effects of the crsA and rvtA suppressors on RNA polymerase and ribosomal protein spots mutations are similar to those previously described for mutations in another intergenic suppressor gene rev. We have examined the effects of rvtA and crsA mutations on the expression of sporulation-associated membrane proteins, including flagellin and penicillin binding protein 5* (PBP 5*). Both suppressors restored sporulation and synthesis of PBP 5* in several spoO mutants. However, only rvtA restored flagellin synthesis in spoO suppressed backgrounds. The membrane protein phenotypes resulting from the presence of crsA or rvtA suppressors in spoO strains suggests that these suppressors function via distinct molecular mechanisms. The rvtA and crsA mutations are also able to block the ability of ethanol to induce spoO phenocopies at concentrations of ethanol which prevent sporulation in wild type cells. The effects of ethanol on sporulation-associated membrane protein synthesis in wild type and suppressor containing strains have been examined.

Isolation and characterization of Bacillus stearothermophilus 30S and 50S ribosomal protein mutations.

Bacillus stearothermophilus mutations which confer resistance to or dependence on a variety of ribosome-targeted antibiotics have been isolated. Many of these mutations produce ribosomal proteins with altered mobilities in a two-dimensional gel electrophoresis system. This collection of altered thermophilic ribosomal proteins will be useful in examining ribosomal structure and function.

The distribution of mutagenic activity in red, rose and white wines.

Using a modified Salmonella typhimurium TA98 Ames-test system, more than 150 red, white and rose wines were analyzed for direct-acting and microsomal enzyme-enhanced mutagenic activity. The following conclusions were reached from analysis of this wine mutagenicity data base. White and rose wines, as well as grape juices, exhibited little or no detectable direct-acting or microsomal enzyme-enhanced mutagenic activity. However, red wine samples contained highly variable amounts of mutagens, ranging from undetectable to levels 30-fold above the sensitivity limit of the assay system. The variations in red wine mutagenicity were unrelated to grape variety, vintage, aging methods or production region. Hence, individual winery production practices must represent the most significant contribution to the variations observed.

Production of an aromatic amino acid auxotroph of a trimethoprim-resistant Escherichia coli and deuteration of the aromatic amino acids in its dihydrofolate reductase.

Interpretation of the 1H-NMR spectra of Escherichia coli dihydrofolate reductase is complicated by the large number of overlapping resonances due to protonated aromatic amino acids. Deuteration of the aromatic protons of aromatic amino acid residues is one technique useful for simplifying the 1H-NMR spectra. Previous attempts to label the dihydrofolate reductase from overproducing strains of Escherichia coli were not completely successful. This labeling problem was solved by transducing via P1 phage a genetic block into the de novo biosynthetic pathway of aromatic amino acids in a trimethoprim resistant strain of E. coli, MB 3746. A new strain, MB 4065, is a very high level producer of dihydrofolate reductase and requires exogenous aromatic amino acids for growth, therefore allowing efficient labeling of its dihydrofolate reductase with exogenous deuterated aromatic amino acid.

Sporulation and the production of serine protease and cephamycin C by Streptomyces lactamdurans.

Streptomyces lactamdurans, producer of the antibiotic cephamycin C, excretes at least two proteases. Physiological studies indicate that antibiotic synthesis and serine protease formation are coordinately regulated. Both are produced only after culture growth ends, and they appear with essentially identical kinetics. In addition, strains which produce superior levels of cephamycin C form equally superior levels of the serine protease. Genetic evidence reveals that the syntheses of the antibiotic and serine proteases are associated with sporulation. Mutants which fail to produce aerial hyphae (bald mutants) also fail to synthesize the antibiotic and serine proteases.

Genetic analysis of Acinetobacter calcoaceticus proline auxotrophs.

Twenty-six Acinetobacter calcoaceticus proline auxotrophs were isolated after ethyl methane sulfonate mutagenesis. Studies using the efficient transformation system of this organism indicate that the mutations comprise therr genetically distinct groups.